Organic compositions to treat EPAS1-related diseases

ABSTRACT

The present disclosure relates to methods of treating EPAS1-related diseases such as cancer, metastases, astrocytoma, bladder cancer, breast cancer, chondrosarcoma, colorectal carcinoma, gastric carcinoma, glioblastoma, head and neck squamous cell carcinoma, hepatocellular carcinoma, lung adenocarcinoma, neuroblastoma, non-small cell lung cancer, melanoma, multiple myeloma, ovarian cancer, rectal cancer, renal cancer, clear cell renal cell carcinoma (and metastases of this and other cancers), gingivitis, psoriasis, Kaposi&#39;s sarcoma-associated herpesvirus, preeclampsia, inflammation, chronic inflammation, neovascular diseases, and rheumatoid arthritis, using a therapeutically effective amount of a RNAi agent to EPAS1.

PRIORITY

This application is divisional of U.S. patent application Ser. No.15/834,323, filed on Dec. 7, 2017, which is a divisional of U.S. patentapplication Ser. No. 14/770,765, filed on Aug. 26, 2015, which is a §371 U.S. National Application of PCT/US2014/018873, filed on Feb. 27,2014, which claims priority to U.S. Provisional Application No.61/770,713, filed on Feb. 28, 2013, the entire contents of which areincorporated herein by reference.

SEQUENCE LISTING

This application contains a Sequence Listing which has been submittedelectronically in ASCII format. The ASCII copy, created on Nov. 27,2017, is named “N054421-US3_SequenceListing.txt” and is 95.6 kb in size.

BACKGROUND OF THE INVENTION

EPAS1 is a member of the HIF (hypoxia inducible factor) gene family.Also known as Hif2 alpha. EPAS1 encodes half of a transcription factorinvolved in the induction of genes regulated by oxygen, and which isinduced as oxygen levels fall (a condition known as hypoxia).

Certain variants of this gene provide protection for people living athigh altitude. However, at low altitude, over-expression of wild-type(WT) EPAS1 is associated with increased hypertension and stroke, andwith symptoms similar to mountain sickness. Mutations in this gene arealso associated with crythroeytosis familial type 4 and pulmonaryhypertension. EPAS1 can also cause excessive production of red bloodcells, leading to inhibited reproductive abilities or even death.

EPAS1 is also required for or enhances the expression of various genesinvolved in an assortment of diseases, including tumor progression. Forexample, EPAS1 plays an important role in the progression of uvealmelanomas, possibly by promoting the autocrine loop VEGF-pVEGFR2/KDR,and by enhancing the expression of LDHA, thus conferring a growthadvantage.

EPAS1 is also involved in or upregulates expression of many factors,including: c-Myc (which favors cell proliferation, transformation,neoplasia and tumorigenesis, and which is highly expressed in mostcancers); Interleukin 8 (a proinflammatory mediator, e.g., in gingivitisand psoriasis): SP-1 (a transcription factor involved in IL-8 regulationand a coactivator of c-Myc); LDH5 (which is linked with tumor necrosisand increased tumor size); and LANA (Latency Associated Nuclear Antigen,which is associated with Kaposi's sarcoma-associated Herpesvirus). Inaddition, HIF (hypoxia induced factor) activity is involved inangiogensis required for cancer tumor growth. EPAS1 is also involved inseveral other diseases, including inflammation, including chronicinflammation, neovascular diseases, rheumatoid arthritis, renal cancer,clear cell renal cell carcinoma (and metastases of this and othercancers), melanoma, uveal melanoma, chondrosarcoma, and multiplemyeloma.

There thus exists the need for treatments related to these and otherEPAS1-related diseases.

BRIEF SUMMARY OF THE INVENTION

The present disclosure encompasses RNAi agents to EPAS1 (Hif2 alpha),for inhibition of EPAS1, and which are useful in treatment ofEPAS1-related diseases, such as cancer, metastases, astrocytoma, bladdercancer, breast cancer, chondrosarcoma, colorectal carcinoma, gastriccarcinoma, glioblastoma, head and neck squamous cell carcinoma,hepatocellular carcinoma, lung adenocarcinoma, neuroblastoma, non-smallcell lung cancer, melanoma, multiple myeloma, ovarian cancer, rectalcancer, renal cancer, clear cell renal cell carcinoma (and metastases ofthis and other cancers), gingivitis, psoriasis, Kaposi'ssarcoma-associated herpesvirus, preeclampsia, inflammation, chronicinflammation, neovascular diseases, and rheumatoid arthritis.

The present disclosure also encompasses a method of treating a humansubject having a pathological state mediated at least in part by EPAS1expression, the method comprising the step of administering to thesubject a therapeutically effective amount of a RNAi agent to EPAS1.

The method also optionally further comprises the step of administering asecond agent. In some aspects, this second agent is another RNAi agentto EPAS1 (e.g., a RNAi agent which targets a different sequence withinthe EPAS1 target). In other aspects, the second agent is anothertreatment, such as one directed to another target, which is alsohyper-active, mutated and/or over-expressed in the pathological state.

The present disclosure provides specific RNAi agents for inhibition ofEPAS1, and methods that are useful in reducing EPAS1 levels in asubject, e.g., a mammal, such as a human. The present disclosurespecifically provides double-stranded RNAi agents comprising at least 15or 19 or more contiguous nucleotides of EPAS1. In particular, thepresent disclosure provides agents comprising sequences of 15 or morecontiguous nucleotides differing by 0, 1, 2 or 3 from those of any ofthe RNAi agents provided, e.g., in any table herein, such as Tables 1 to5 (including all parts of Table 5, from Table 5A to 5E). The RNAi agentsparticularly can in one aspect comprise less than 30 nucleotides perstrand, e.g., such as 17-23 nucleotides, 15-19, 18-22, and/or 19-21nucleotides, and/or such as those provided, e.g., in Tables 1 to 5, andmodified and unmodified variants thereof (e.g., wherein the sense and/oranti-sense or first and/or second strand are modified or unmodified).The present disclosure also provides agents comprising a sense strandand an anti-sense strand, wherein the sense and/or the anti-sense strandcomprise sequences of 19 or more contiguous nucleotides differing by 0,1, 2 or 3 from those of the RNAi agents provided, e.g., in Tables 1 to5, and modified or unmodified variants thereof. The sense and anti-sensestrand can be contiguous, or physically connected, e.g., by covalentlybonds, a loop or tinker.

The double-stranded RNAi agents can have 0, 1 or 2 blunt ends, and/oroverhangs of 1, 2, 3 or 4 nucleotides (i.e., 1 to 4 nt) from one or both3′ and/or 5′ ends. The double-stranded RNAi agents can also optionallycomprise one or two 3′ caps and/or one or more modified nucleotides.Modified variants of sequences as provided herein include those that areotherwise identical but contain substitutions of a naturally-occurringnucleotide for a corresponding modified nucleotide.

Furthermore, the RNAi agent can either contain only naturally-occurringribonucleotide subunits, or one or more modifications to the sugar,phosphate or base of one or more of the replacement nucleotide subunits,whether they comprise ribonucleotide subunits or deoxyribonucleotidesubunits. In one aspect, modified variants of the disclosed RNAi agentshave a thymidine (as RNA, or, preferably, DNA) replacing a uridine, orhave an inosine base. In some aspects, the modified variants of thedisclosed RNAi agents can have a nick in the passenger strand,mismatches between the guide and passenger strand, DNA replacing the RNAof a portion of both the guide and passenger strand (e.g., the seedregion), and/or a shortened passenger strand (e.g., 13, 14, 15, 16, 17or 18 nt). Once a functional guide strand is identified, modificationsand variants of the RNAi agent can be readily made. Any two or moremodifications which are not mutually exclusive can be combined (e.g.,the combination of base modifications with shortened passenger strand;or nicked passenger strand and base modifications; or DNA replacing partor all of the seed region and base modifications in the remaining RNA;etc.). Any sequence or any portion thereof (e.g., 19 [or more]contiguous nt; 15 [or more] contiguous nt; 15 [or more] contiguous ntdiffering by 0, 1, 2 or 3 nt) disclosed herein can be used with anymodification or modification scheme disclosed herein, provided they arenot mutually exclusive (e.g., refer to different lengths of strands).The sequences (and any portions thereof) of RNAi agents can be used withany modification, set of modifications (e.g., modification scheme),vehicle, composition, method, treatment, etc., described herein,provided they are not mutually exclusive.

In one aspect, modified variants of the disclosed RNAi agents includeRNAi agents with the same sequence (e.g., the same sequence of bases) asany RNAi agent disclosed in any of Tables 1 to 5, but with one or moremodifications to one or more of the sugar or phosphate of one or more ofthe nucleotide subunits. In one aspect, the modifications improveefficacy, stability (e.g., against nucleases in, for example, bloodserum or intestinal fluid), and/or reduce immunogenicity of the RNAiagent. One aspect of the present disclosure relates to a double-strandedoligonucleotide comprising at least one non-natural nucleobaxe. Theseinclude universal base analogues, e.g., those described by Loakes 2001Nucl. Acids Res. 29: 2437-2447. In certain aspects, the non-naturalnucleobase is difluorotolyl, nitroindolyl, nitropyrrolyl, ornitroimidazolyl. In a particular aspect, the non-natural nucleobase isdifluorotolyl. In certain aspects, only one of the two oligonucleotidestrands contains a non-natural nucleobase. In certain aspects, both ofthe oligonucleotide strands contain a non-natural nucleobase.

The RNAi agent(s) can optionally be attached to a ligand selected toimprove one or more characteristic, such as, e.g., stability,distribution and/or cellular uptake of the agent, e.g., cholesterol or aderivative thereof. The RNAi agent(s) can be isolated or be part of apharmaceutical composition used for the methods described herein.Particularly, the pharmaceutical composition can be formulated fordelivery to specific tissues (e.g., those afflicted with a EPAS1-relateddisease) or formulated for parenteral administration. The pharmaceuticalcomposition can optionally comprise two or more RNAi agents, each onedirected to the same, overlapping or a different segment of the EPAS1mRNA. Optionally, the pharmaceutical composition can further comprise orbe used in conjunction with any known treatment for any EPAS1-relateddisease.

The present disclosure further provides methods for reducing the levelof EPAS1 mRNA in a cell, particularly in the case of a diseasecharacterized by over-expression or hyper-activity of EPAS1. Cellscomprising an alteration such as a mutation, over-expression and/orhyperactivity of EPAS1 are termed “EPAS1-defective” cells. Such methodscomprise the step of administering one or more of the RNAi agents of thepresent disclosure to an EPAS1-defective cell, as further describedbelow. The present methods utilize the cellular mechanisms involved inRNA interference to selectively degrade the target RNA in a cell and arecomprised of the step of contacting a cell with one of the RNAi agentsof the present disclosure.

The present disclosure also encompasses a method of treating a humansubject having a pathological state mediated at least in part by EPAS1expression, the method comprising the step of administering to thesubject a therapeutically effective amount of a RNAi agent EPAS1.Additional methods involve preventing, treating, modulating and/orameliorating a pathological state wherein disease progression (e.g.,tumor growth) requires EPAS1, although EPAS1 is not amplified orover-expressed. Such methods comprise the step of administering one ofthe RNAi agents of the present disclosure to a subject, as furtherdescribed below. Such methods can be performed directly on a cell or canbe performed on a mammalian subject by administering to a subject one ofthe RNAi agents' pharmaceutical compositions of the present disclosure.Reduction of target EPAS1 RNA in a cell results in a reduction in theamount of encoded EPAS1 protein produced. In an organism, this canresult in restoration of balance in a pathway involving EPAS1, and/orprevention of EPAS1 accumulation, and/or a reduction in EPAS1 activityand/or expression, and/or prevention of EPAS1-mediated activation ofother genes, and/or amelioration, treatment and/or prevention of aEPAS1-related disease. In one aspect, a reduction in EPAS1 expression,level or activity can limit tumor growth.

The methods and compositions of the present disclosure, e.g., themethods and EPAS1 RNAi agent compositions, can be used in anyappropriate dosage and/or formulation described herein or known in theart, as well as with any suitable route of administration describedherein or known in the art.

The details of one or more aspects of the present disclosure are setforth in the accompanying drawings and the description below. Elementsof the various aspects (e.g., sequences or portions thereof [e.g., 15 ormore contiguous nt differing by 0, 1, 2 or 3 nt], lengths,modifications, terminal dinucleotides, endcaps, combinations of RNAiagents, combination therapy involving a EPAS1 RNAi agent and anotheragent, conjugation with other components, compositions or methods ortechniques for delivery, disease treatment, etc.) disclosed herein orknown in the art which are not mutually exclusive can be combined witheach other, provided that the agent or agents are still capable ofmediating RNA interference. For example, any RNAi agent sequencedisclosed herein can be combined with any set of modifications orendcaps disclosed herein. Similarly, any combination of modifications,5′ end caps, and/or 3′ end caps can be used with any RNAi agent sequencedisclosed herein. Any RNAi agent disclosed herein (with any combinationof modifications or endcaps or without either modifications or endcaps)can be combined with any other RNAi agent or other treatment compositionor method disclosed herein.

Other features, objects, and advantages of the present disclosure willbe apparent from this description, the drawings, and from the claims.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 illustrates various example modified nucleotides which have beenor can be used in modified variants of EPAS1 RNAi agents: U002, U003,U004, U005, C004, C005, A004, A005, G005, and G004, which can be used inthe RNAi agents disclosed herein. U002 indicates a 2′-deoxy-thymidinewhich is DNA. U003 indicates 2′-deoxy uridine. U004 indicates anucleotide with a Uridine (“U”) base with a 2′-O-methyl modification.U005 indicates a U base with a 2′-O-methoxyethyl (MOE) modification.C004 indicates a Cytosine (“C”) base with a 2′-O-methyl modification.C005 indicates a C base with 2′-O-methoxyethyl modification. A004indicates an Adenosine (“A”) base with a 2′-O-methyl modification. A005indicates an A base with 2′-O-methoxyethyl modification. G005 indicatesa Guanosine (“G”) base with a 2′O-methyl modification. G004 indicates aG base with a 2′O-methyl modification.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure encompasses RNAi agents to EPAS1, for targetingand inhibition of EPAS1, which are useful in treatment of EPAS1-relateddiseases (e.g., diseases associated with mutations in and/or alteredexpression, level and/or activity of EPAS1, diseases requiring EPAS1,diseases affected by a factor whose expression, over-expression, orhyper-activity is directly or indirectly affected by EPAS1, and/ordiseases treatable by modulating the expression, level and/or activityof EPAS1. Such EPAS1-related diseases include; cancer, metastases,astrocytoma, bladder cancer, breast cancer, chondrosarcoma, colorectalcarcinoma, gastric carcinoma, glioblastoma, head and neck squamous cellcarcinoma, hepatocellular carcinoma, lung adenocarcinoma, neuroblastoma,non-small cell lung cancer, melanoma, multiple myeloma, ovarian cancer,rectal cancer, renal cancer, clear cell renal cell carcinoma (andmetastases of this and other cancers), gingivitis, psoriasis. Kaposi'ssarcoma-associated herpesvirus, preeclampsia, inflammation, chronicinflammation, neovascular diseases, and rheumatoid arthritis. Thepresent disclosure also provides methods of treating a human subjecthaving a pathological state mediated at least in part by EPAS1expression or over-expression or hyper-activity, or requiring EPAS1, themethod comprising the step of administering to the subject atherapeutically effective amount of a RNAi agent to EPAS1.

Various Aspects of the Disclosure Include the Following.

An RNAi Agent Comprising an Antisense Strand of an RNAi Agent DescribedHerein.

In one aspect, an aspect of the present disclosure relates to acomposition comprising an RNAi agent comprising an antisense strand,wherein the antisense strand comprises at least 15 contiguousnucleotides (nt) differing by 0, 1, 2, or 3 nucleotides from theantisense strand of an RNAi agent to EPAS1 selected from any sequenceprovided herein (e.g., in any one or more of Tables 1 to 5, etc.). Inanother aspect, the present disclosure relates to a compositioncomprising an RNAi agent comprising a first strand and a second strand,wherein the first strand comprises at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nucleotides from the first strand of an RNAiagent to EPAS1 from any sequence provided herein, in another aspect, thepresent disclosure relates to a composition comprising an RNAi agentcomprising a sense strand and an anti sense strand, wherein the sensestrand comprises at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nucleotides from the sense strand and the antisense strandcomprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3nucleotides from the antisense strand of an RNAi agent to EPAS1 listedimmediately above. In another aspect, the present disclosure relates toa composition comprising an RNAi agent comprising a first strand and asecond strand, wherein the sequence of the first strand comprises thesequence of the first strand of any sequence provided herein. In anotheraspect, the present disclosure relates to a composition comprising anRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is the sequence of the first strand of anysequence provided herein. In various aspects, the first and secondstrands are the anti-sense and sense strand, respectively, of any RNAiagent disclosed herein. In various aspects, the first and second strandsare the sense and anti-sense strand, respectively, of any RNAi agentdisclosed herein.

Particular duplexes include the unmodified (e.g., “generic”) and examplemodified variants listed in Table 1; additional sequences and data forthese RNAi agents are presented in the subsequent Tables, in addition tothe described example modifications, other modified variants can be madeusing the nucleotide sequences provided. In various aspects, the firstand/or second strand are modified or unmodified.

Tables

Provided in Table 1 are the names of EPAS1 RNAi agents (derived fromtheir position in the human EPAS1 gene sequence NM 001430), along withthe SEQ ID NOs. representing the DNA sequence, unmodified sequence, andexample modified sequences (with both A51 S26 and A85 S26 examplemodified sequences). Both sense and antisense (AS) sequences arepresented in these Tables.

Table 2 provides the DNA sequences.

Table 3 provides the unmodified sequences of the RNAi agents.

Table 4 indicates whether or not particular RNAi agent sequences (whichare derived from human) also correspond to those from mouse (Mu or mm),rat (Ra or m), and Rhesus [mmu or Macaca mulatta].

Table 5 (comprising Tables 5A to 5E) list example modified RNAi agentsequences.

Tables 6 to 8 show efficacy of various EPAS1 RNAi agents in vitro and invivo.

Table 9 shows overlapping groups of these EPAS1 RNAi agents.

TABLE 1 SEQ ID NOs for various EPAS1 RNAi agents. Presented are the SEQID NOs. representing the DNA sequence, unmodified sequence, and examplemodified sequences (with both A51 S26 and A85 S26 example modifiedsequences). Unmodified RNAi Example Example agent Modified Modified DNAAnti- (A51 S26) (A85 S26) Name Sense AS Sense sense SENSE AS Sense ASPosition SEQ SEQ SEQ SEQ SEQ SEQ SEQ SEQ NM001430 ID NO: ID NO: ID NO:ID NO: ID NO: ID NO: ID NO: ID NO: 842 1 20 39 58 145 126 77 96 2802 221 40 59 146 127 78 97 3040 3 22 41 60 147 128 79 98 3304 4 23 42 61 148129 80 99 3310 5 24 43 62 149 130 81 100 3345 6 25 44 63 150 131 82 1013354 7 26 45 64 151 132 83 102 3735 8 27 46 65 152 133 84 103 3739 9 2847 66 153 134 85 104 3875 10 29 48 67 154 135 86 105 4153 11 30 49 68155 136 87 106 4157 12 31 50 69 156 137 88 107 5049 13 32 51 70 157 13889 108 5057 14 33 52 71 158 139 90 109 5058 15 34 53 72 159 140 91 1105059 16 35 54 73 160 141 92 111 5108 17 36 55 74 161 142 93 112 5144 1837 56 75 162 143 94 113 5149 19 38 57 76 163 144 95 114Additional modified variants of EPAS1 RNAi agents are shown in Tables 5Cto 5E.

Table 1 thus presents the SEQ ID NO identifiers of the sense andanti-sense strands of unmodified and an example modified EPAS1 RNAiagents. For example, in this Table, the unmodified sense and anti-sensesequences of RNAi agent 842 are represented by SEQ ID NOs.: 39 and 58,respectively. A modified variant of this RNAi agent is represented bySEQ ID NOs: 145 and 126; another example modified variant is representedby SEQ ID NOs: 77 and 96. Also note that the name (e.g., 842) is derivedfrom the position number in the human EPAS1 gene sequence NM 001430.These names are sometimes prefixed with “EPAS1” or “EPAS1_” (e.g.,“EPAS1 842” or EPAS1_842”). Also note that the prefix “AD” is onoccasion replaced by “ND”. The name of the RNAi agent also sometimes hasa suffix, such as .1. This indicates a particular variant. Thus, “842”and “EPAS1_842” and the like all indicate RNAi agents of the samesequence, although they may differ in modifications.

In the sequences in Tables 5, lower-case letters (e.g., c, u) indicatemodified nucleotides while upper ease letters (e.g., C, U, A, G)indicate unmodified nucleotides. In this Table, example modifiedversions of each of the sequences are shown. However, the presentdisclosure also contemplates and encompasses unmodified versions ofthese sequences and other versions which comprise additional oralternative modifications.

In the sequences in the Tables, the modified and unmodified variants canoptionally further comprise the sequence “TT”, “dTdT”, “dTsdT” or “UU”as a single-stranded overhang at the 3′ end, also termed herein aterminal dinucleotide or 3′ terminal dinucleotide. dT is2′-deoxy-thymidine-5′-phosphate and sdT is 2′-deoxy Thymidine5′-phosphorothioate. In the disclosed sequences, terminal dinucleotide“UU” is UU or 2′-OMe-U 2′-OMe-U, and the terminal TT and the terminal UUcan be in the inverted/reverse orientation. The terminal dinucleotide(e.g., UU) is not part of the EPAS1 target sequence, but is a modifiedvariant of the dithymidine dinucleotide commonly placed as an overhangto protect the ends of siRNAs from nucleases (see, for example, Elbashiret al. 2001 Nature 411: 494-498; Elbashir et al. 2001 EMBO J. 20:6877-6888; and Kraynack et al. 2006 RNA 12:163-176). A terminaldinucleotide is known from these references to enhance nucleaseresistance but not contribute to target recognition. Thus, the presentdisclosure also encompasses any modified or any unmodified variantdisclosed herein, wherein the modified variant comprises a terminal TT,dTdT, sdT, dTsdT, sdTsdT, sdTdT, or the like which may be in either theinverted/reverse orientation or in the same 5′ to 3′ orientation as theEPAS1 specific sequence in the duplex. In addition, terminology usedherein referring to “the EPAS1 portion of a RNAi agent sequence” and thelike indicate the portion of the sequence of a RNAi agent which isderived from EPAS1 (thus “the EPAS1 portion of a RNAi agent sequence”does not include, for example, a terminal dTdT, TT, UU, U (2′-OMe) dT. U(2′-OMe) U (2′-OMe), T (2′-OMe), T (2′-OMe), T(2′-OMe) dT, or the like,but does include the portion of the RNAi agent that corresponds to or iscomplementary to a portion of the EPAS1 gene sequence or mRNA sequence.In one aspect, the composition comprises a RNAi agent comprising a firstand an second strand, wherein the sequence of the first strand and thesequence of the second strand are the sequences of the first and secondstrand, respectively, of any RNAi agent provided herein, furthercomprising a 3′ terminal dinucleotide (a single-stranded overhangcomprising 2 nt at the 3′ end). In one aspect, the composition comprisesa RNAi agent comprising a first and an second strand, wherein thesequence of the first strand and the sequence of the second strand arethe sequences of the first and second strand, respectively, of any RNAiagent provided herein, further comprising a 3′ terminal dinucleotideselected from TT, UU, U (2′-OMe) dT, U (2′-OMe) U (2′-OMe), T(2′-OMe) T(2′-OMe), T(2′-OMe) dT, dTdT, sdT, dTsdT, sdTsdT, and sdTdT. In oneaspect, the composition comprises a RNAi agent comprising a first and ansecond strand, wherein the sequence of the first strand and the sequenceof the second strand are the sequences of the first and second strand,respectively, of any RNAi agent provided herein, further comprising a 3′terminal UU dinucleotide.

On any modified or unmodified variant, a 3′ end cap, as is known in theart, can be used instead of or in addition to a terminal dinucleotide tostabilize the end from nuclease degradation provided that the 3′ end capis able to both stabilize the RNAi agent (e.g., against nucleases) andnot interfere excessively with siRNA activity. Thus, the presentdisclosure also encompasses any modified or any unmodified variantdisclosed herein, wherein the modified variant further comprises aterminal 3′ end cap.

An RNAi Agent Comprising an Antisense Strand of an RNAi Agent DescribedHerein.

In one particular specific aspect, the present disclosure relates to acomposition comprising an RNAi agent comprising an antisense strand,wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of an RNAi agent to EPAS1 selected from those antisense strandsin the specific duplexes provided above and as listed in Table 1.

Various particular specific aspects of this aspect are described below.

In one aspect, the composition further comprises a second RNAi agent toEPAS1. In various aspects, the second RNAi agent is physically separatefrom the first, or the two are physically connected (e.g., covalentlylinked or otherwise conjugated).

In one aspect, the composition comprises a RNAi agent comprising a firstand an second strand, wherein the sequence of the first strand and thesequence of the second strand are the sequences of the first and secondstrand, respectively, of any RNAi agent provided herein. In one aspect,the composition comprises a RNAi agent comprising a first and an secondstrand, wherein the sequence of the first strand and the sequence of thesecond strand are the sequences of the first and second strand,respectively, of any RNAi agent provided herein, further comprising anadditional about 6 to 20 nucleotides on one or both strands (e.g., about6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nt). In oneaspect, the composition comprises a RNAi agent comprising a first and ansecond strand, wherein the sequence of the first strand and the sequenceof the second strand are the sequences of the first and second strand,respectively, of any RNAi agent provided herein, further comprising a 3′terminal dinucleotide. In one aspect, the composition comprises a RNAiagent comprising a first and an second strand, wherein the sequence ofthe first strand and the sequence of the second strand are the sequencesof the first and second strand, respectively, of any RNAi agent providedherein, further comprising a 3′ terminal dinucleotide selected from TT,UU, U (2′-OMe) dT, U (2′-OMe) U (2′-OMe), T(2′-OMe) T (2′-OMe),T(2′-OMe) dT, dTdT, sdT, dTsdT, sdTsdT, and sdTdT. In one aspect, thecomposition comprises a RNAi agent comprising a first and an secondstrand, wherein the sequence of the first strand and the sequence of thesecond strand are the sequences of the first and second strand,respectively, of any RNAi agent provided herein, further comprising a 3′terminal UU dinucleotide. The various aspects, the first and secondstrands are the sense and anti-sense strands listed in the Tablesherein, respectively. In various aspects, the first and second strandsare the anti-sense and sense strands listed in the Tables herein,respectively.

In one aspect, the sense strand is about 30 or fewer nucleotides (nt) inlength.

In one aspect, the antisense strand is about 30 or fewer nucleotides inlength.

In one aspect, the antisense strand forms a duplex region with a sensestrand, wherein the duplex region is about 15 to 30 nucleotide pairs inlength.

In one aspect, the antisense strand is about 15 to about 30 nucleotidesin length, including about 19 to about 23 nucleotides in length. In oneaspect, the antisense strand has at least the length selected from about15 nucleotides, about 16 nucleotides, about 17 nucleotides, about 18nucleotides, about 19 nucleotides, about 20 nucleotides, about 21nucleotides, about 22 nucleotides, about 23 nucleotides, about 24nucleotides, about 25 nucleotides, about 26 nucleotides, about 27nucleotides, about 28 nucleotides, about 29 nucleotides and 30nucleotides.

In one aspect, the RNAi agent comprises a modification that causes theRNAi agent to have increased stability in a biological sample orenvironment (e.g., cytoplasm, interstitial fluid, blood serum, or lungor intestinal lavage).

In one aspect, the RNAi agent comprises at least one sugar backbonemodification (e.g., phosphorothioate linkage) or at least one2′-modified nucleotide.

In one aspect, the RNAi agent comprises: at least one5′-uridine-adenine-3′ (5′-ua-3′) dinucleotide, wherein the uridine is a2′-modified nucleotide; at least one 5′-uridine-guanine-3′ (5′-ug-3′)dinucleotide, wherein the 5′-uridine is a 2′-modified nucleotide; atleast one 5′-cytidine-adenine-3′ (5′-ca-3′) dinucleotide, wherein the5′-cytidine is a 2′-modified nucleotide; or at least one5′-uridine-uridine-3′ (5′-uu-3′) dinucleotide, wherein the 5′-uridine isa 2′-modified nucleotide. These dinucleotide motifs are particularlyprone to serum nuclease degradation (e.g. RNase A). Chemicalmodification at the 2′-position of the first pyrimidine nucleotide inthe motif prevents or slows down such cleavage. This modification recipeis also known under the term ‘endo light’.

In one aspect, the RNAi agent comprises a 2′-modification selected fromthe group consisting of: 2′-deoxy, 2′-deoxy-2′-fluoro. 2′-O-methyl(2′-OMe or 2′OMe), 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl(2′-O-AP). 2′-O-dimethylaminoethyl (2′-O-DMAOE),2′-O-dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl(2′-O-DMAEOE), and 2′-O—N-methylacetamido (2′-O-NMA). In one aspect, allpyrimidines (uridine and cytidine) are 2′-O-methyl-modified nucleotides.

In another aspect, the RNAi agent comprises a 2′-modification selectedfrom the group consisting of:

In one aspect, the RNAi agent comprises at least one blunt end.

In one aspect, the RNAi agent comprises an overhang having 1 nt to 4 ntunpaired.

In one aspect, the RNAi agent comprises an overhang at the 3′-end of theantisense strand of the RNAi agent.

In one aspect, the RNAi agent is ligated to one or more diagnosticcompound, reporter group, cross-linking agent, nuclease-resistanceconferring moiety, natural or unusual nucleobase, lipophilic molecule,cholesterol, lipid, lectin, steroid, uvaol, hecigenin, diosgenin,terpene, triterpene, sarsasapogenin, Friedelin,epifriedelanol-derivatized lithocholic acid, vitamin, carbohydrate,dextran, pullulan, chitin, chitosan, synthetic carbohydrate, oligolactate 15-mer, natural polymer, low- or medium-molecular weightpolymer, inulin, cyclodextrin, hyaluronic acid, protein, protein-bindingagent, integrin-targeting molecule, polycationic, peptide, polyamine,peptide mimic, and/or transferrin.

In one aspect, the RNAi agent is capable of inhibiting expression ofEPAS1 by at least about 50% in 786-O tumors in nude mice.

In one aspect, the RNAi agent is capable of inhibiting expression ofEPAS1 by at least about 70% at a concentration of 10 nM in 786-O cellsin vitro.

In one aspect, the RNAi agent is capable of inhibiting expression ofEPAS1 by at least about 75% at a concentration of 10 nM in 786-O cellsin vitro.

In one aspect, the RNAi agent is capable of inhibiting expression ofEPAS1 by at least about 80% at a concentration of 10 nM in 786-O cellsin vitro.

In one aspect, the RNAi agent is capable of inhibiting expression ofEPAS1 by at least about 90% at a concentration of 10 nM in 786-O cellsin vitro.

In one aspect, the RNAi agent is capable of inhibiting expression ofEPAS1 by at least about 95% at a concentration of 10 nM in 786-O cellsin vitro.

In one aspect, the RNAi agent is capable of inhibiting expression ofEPAS1 by at least about 99% at a concentration of 10 nM in 786-O cellsin vitro.

In one aspect, the RNAi has an EC50 of no more than about 0.1 nM in786-O cells in vitro. EC50 is effective concentration to reduce geneexpression by 50%.

In one aspect, the RNAi has an EC50 of no more than about 0.01 nM in786-O cells in vitro.

In one aspect, the RNAi has an EC50 of no more than about 0.001 nM in786-O cells in vitro.

An RNAi Agent Comprising a Sense and Antisense Strand of an RNAiDescribed Herein.

In one particular specific aspect, the present disclosure relates to acomposition comprising a RNAi agent comprising a first strand and asecond strand, wherein the sequence of the first strand and/or secondstrand comprise at least 15 contiguous nucleotides, differing by 0, 1,2, or 3 nucleotides from the sequence of the first and/or second strandof a RNAi agent to EPAS1 selected from the specific duplexes providedherein and listed, e.g., in any Table herein. In one particular specificaspect, the present disclosure relates to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand and/or second strand comprise at least 15contiguous nucleotides from the sequence of the first and/or secondstrand of a RNAi agent to EPAS1 selected from the specific duplexesprovided herein and listed, e.g., in any Table herein. In one particularspecific aspect, the present disclosure relates to a compositioncomprising a RNAi agent comprising a first strand and a second strand,wherein the sequence of the first strand and/or second strand compriseat least 19 contiguous nucleotides (e.g., nt 1-19, nt 2-20, nt 3-21,etc.) from the sequence of the first and/or second strand of a RNAiagent to EPAS1 selected from the specific duplexes provided herein andlisted, e.g., in any Table herein.

In one particular specific aspect, the present disclosure relates to acomposition comprising a RNAi agent comprising a first strand and asecond strand, wherein the sequence of the first strand and/or secondstrand comprise the sequence of the first and/or second strand,respectively, of a RNAi agent to EPAS1 selected from the specificduplexes provided herein and listed, e.g., in any Table herein.

In one particular specific aspect, the present disclosure relates to acomposition comprising a RNAi agent comprising a first strand and asecond strand, wherein the sequence of the first strand and/or secondstrand are the sequence of the first and/or second strand, respectively,of a RNAi agent to EPAS1 selected from the specific duplexes providedherein and listed, e.g., in any Table herein.

In one particular specific aspect, the present disclosure relates to acomposition comprising a RNAi agent comprising a first strand and asecond strand, wherein the sequence of the first strand and/or secondstrand are the sequence of the first and/or second strand, respectively,of a RNAi agent to EPAS1 selected from the specific duplexes providedherein and listed, e.g., in any Table herein, wherein the sequence ofthe first and/or second strand further comprise a terminal dinucleotide.

In one particular specific aspect, the present disclosure relates to acomposition comprising a RNAi agent comprising a first strand and asecond strand, wherein the sequence of the first strand and/or secondstrand are the sequence of the first and/or second strand, respectively,of a RNAi agent to EPAS1 selected from the specific duplexes providedherein and listed, e.g., in any Table herein, wherein the sequence ofthe first and/or second strand further comprise a terminal UUdinucleotide.

In one particular specific aspect, the present disclosure relates to acomposition comprising an RNAi agent comprising a sense strand and anantisense strand, wherein the sense strand and antisense strand compriseat least 15 contiguous nucleotides differing by 0, 1, 2, or 3nucleotides, from the sense and antisense strand, respectively, of anRNAi agent to EPAS1 selected from the specific duplexes provided aboveand as listed in Table 1. In one particular specific aspect, the presentdisclosure relates to a composition comprising an RNAi agent comprisinga sense strand and an antisense strand, wherein the sense strand andantisense strand comprise at least 15 contiguous nucleotides from thesense and antisense strand, respectively, of an RNAi agent to EPAS1selected from the specific duplexes provided above and as listed inTable 1. In one particular specific aspect, the present disclosurerelates to a composition comprising an RNAi agent comprising a sensestrand and an antisense strand, wherein the sense strand and antisensestrand comprise at least 19 contiguous nucleotides (e.g., nt 1-19, nt2-20, or nt 3-21) from the sense and antisense strand, respectively, ofan RNAi agent to EPAS1 selected from the specific duplexes providedabove and as listed in Table 1.

Various particular specific aspects of this aspect are described below.

In one aspect, the composition comprises a second RNAi agent to EPAS1.In various aspects, the second RNAi agent is physically separate fromthe first, or the two are physically connected (e.g., chemically linkedor otherwise conjugated). In some aspects, the first and second RNAiagents are combined within the same composition (e.g., both in the samelipid nanoparticle).

In one aspect, the antisense strand is about 30 or fewer nucleotides inlength.

In one aspect, the sense strand and the antisense strand form a duplexregion about 15 to about 30 nucleotide pairs in length.

In one aspect, the antisense strand is about 15 to about 36 nt in lengthincluding about 18 to about 23 nt in length, and including about 19 toabout 21 nt in length and about 19 to about 23 nt in length. In oneaspect, the antisense strand has at least the length selected from about15 nt, about 16 nt, about 17 nt, about 18 nt, about 19 nt, about 20 nt,about 21 nt, about 22 nt, about 23 nt, about 24 nt, about 25 nt, about26 nt, about 27 nt, about 28 nt, about 29 nt and about 30 nt.

In one aspect, the RNAi agent comprises a modification that causes theRNAi agent to have increased stability in a biological sample orenvironment.

In one aspect, the RNAi agent comprises a modified sugar backbone suchas, e.g., a phosphorothioate linkage, or comprises a 2′-modifiednucleotide.

In one aspect, the RNAi agent comprises: at least one5′-uridine-adenine-3′ (5′-ua-3′) dinucleotide, wherein the uridine is a2′-modified nucleotide; at least one 5′-uridine-guanine-3′ (5′-ug-3′)dinucleotide, wherein the 5′-uridine is a 2′-modified nucleotide; atleast one 5′-cytidine-adenine-3′ (5′-ca-3′) dinucleotide, wherein the5′-cytidine is a 2′-modified nucleotide; or at least one5′-uridine-uridine-3′ (5′-uu-3′) dinucleotide, wherein the 5′-uridine isa 2′-modified nucleotide.

In one aspect, the RNAi agent comprises a 2′-modification selected fromthe group consisting of: 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-methyl,2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE). 2′-O-dimethylaminopropyl(2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), and2′-O—N-methylacetamido (2′-O-NMA). In one aspect, all pyrimidines(uridine and cytidine) are 2′-O-methyl-modified nucleotides.

In one aspect, the RNAi agent comprises at least one blunt end.

In one aspect, the RNAi agent comprises an overhang having 1 to 4 ntunpaired.

In one aspect, the RNAi agent comprises an overhang at the 3′-end of theantisense strand of the RNAi agent.

In one aspect, the RNAi agent is ligated to one or more diagnosticcompound, reporter group, cross-linking agent, nucleuse-resistanceconferring moiety, natural or unusual nucleobase, lipophilic molecule,cholesterol, lipid, lectin, steroid, uvaol, hecigenin, diosgenin,terpene, triterpene, sarsasapogenin, Friedelin,epifriedelanol-derivatized lithocholic acid, vitamin, carbohydrate,dextran, pullulan, chitin, chitosan, synthetic carbohydrate, oligolactate 15-mer, natural polymer, low- or medium-molecular weightpolymer, inulin, cyclodextrin, hyaluronic acid, protein, protein-bindingagent, integrin-targeting molecule, polycationic, peptide, polyamine,peptide mimic, and/or transferrin.

In one aspect, the RNAi agent is capable of inhibiting expression ofEPAS1 by at least about 50% in 786-O tumors in nude mice.

In one aspect, the RNAi agent is capable of inhibiting expression ofEPAS1 by at least about 70% at a concentration of 10 nM in 786-O cellsin vitro.

In one aspect, the RNAi agent is capable of inhibiting expression ofEPAS1 by at least about 80% at a concentration of 10 nM in 786-O cellsin vitro.

In one aspect, the RNAi agent is capable of inhibiting expression ofEPAS1 by at least about 90% at a concentration of 10 nM in 786-O cellsin vitro.

In one aspect, the RNAi agent is capable of inhibiting expression ofEPAS1 by at least about 95% at a concentration of 10 nM in 786-O cellsin vitro.

In one aspect, the RNAi agent is capable of inhibiting expression ofEPAS1 by at least about 99% at a concentration of 10 nM in 786-O cellsin vitro.

In one aspect, the RNAi has an EC50 of no more than about 0.1 nM in786-O cells in vitro.

In one aspect, the RNAi has an EC50 of no more than about 0.01 nM in786-O cells in vitro.

In one aspect, the RNAi has an EC50 of no more than about 0.001 nM in786-O cells in vitro.

A Method of Treatment Using a Composition Comprising a RNAi AgentDescribed Herein.

In one particular specific aspect, the present disclosure relates to amethod of treating a EPAS1-related disease in an individual, comprisingthe step of administering to the individual a therapeutically effectiveamount of a composition comprising an RNAi agent comprising an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of an RNAi agent to EPAS1 selected from those specific duplexesprovided above and as listed in Table 1. In one aspect, the RNAi agentto EPAS1 comprises an antisense strand duplexed with a sense strand,wherein the sense and antisense strands are selected from one or more ofthe sequences provided in any of Tables 1 to 5.

Various particular specific aspects of this aspect are described below.Any aspects disclosed herein that are not mutually exclusive can becombined.

In one aspect, the present disclosure relates to such a method, whereinthe composition comprising a RNAi agent further comprises a Sensestrand, wherein the sense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the sense strandof a RNAi agent to EPAS1 selected from the specific duplexes providedherein and as listed, e.g., in any Table herein.

In one aspect of the method, the RNAi agent comprises at least ananti-sense strand, and/or comprises a sense and an anti-sense strand,wherein the sequence of the sense and/or anti-sense strand is thesequence of the sense and/or the anti-sense strand of a RNAi agent toEPAS1 selected from those specific duplex provided herein and as listed,e.g., in Table 1, wherein the composition further comprises apharmaceutically effective formulation.

In one aspect of the method, the RNAi agent comprises at least ananti-sense strand, and/or comprises a sense and an anti-sense strand,wherein the sequence of the sense and/or anti-sense strand comprises thesequence of the sense and/or the anti-sense strand of a RNAi agent toEPAS1 selected from those specific duplex provided herein and as listed,e.g., in Table 1, wherein the composition further comprises apharmaceutically effective formulation.

In one aspect, the EPAS1-related disease is cancer, metastases,astrocytoma, bladder cancer, breast cancer, chondrosarcoma, colorectalcarcinoma, gastric carcinoma, glioblastoma, head and neck squamous cellcarcinoma, hepatocellular carcinoma, lung adenocarcinoma, neuroblastoma,non-small cell lung cancer, melanoma, multiple myeloma, ovarian cancer,rectal cancer, renal cancer, clear cell renal cell carcinoma (andmetastases of this, and other cancers), gingivitis, psoriasis, Kaposi'ssarcoma-associated herpesvirus, preeclampsia, inflammation, chronicinflammation, neovascular diseases, and rheumatoid arthritis.

In one aspect, the EPAS1-related disease is cancer.

In one aspect, the method further comprises the step of administering anadditional treatment.

In one aspect, the additional treatment is a method (or procedure). Inone aspect, the additional treatment is a therapeutically effective doseof a composition.

In one aspect, the additional treatment and the RNAi agent can beadministered in any order, or can be administered simultaneously.

In one aspect, the method further comprises the step of administering anadditional treatment for cancer, metastases, astrocytoma, bladdercancer, breast cancer, chondrosarcoma, colorectal carcinoma, gastriccarcinoma, glioblastoma, head and neck squamous cell carcinoma,hepatocellular carcinoma, lung adenocarcinoma, neuroblastoma, non-smallcell lung cancer, melanoma, multiple myeloma, ovarian cancer, rectalcancer, renal cancer, clear cell renal cell carcinoma (and metastases ofthis and other cancers), gingivitis, psoriasis, Kaposi'ssarcoma-associated herpesvirus, preeclampsia inflammation, chromicinflammation, neovascular diseases, and rheumatoid arthritis.

In one aspect, the method further comprises the step of administering anadditional treatment. A RNAi agent to EPAS1 can be used in conjunctionwith any additional treatment disclosed herein, as appropriate for thedisease, optionally, in further conjunction with one or more additionalRNAi agents to EPAS1.

It will be understood that references to any additional treatment aremeant to also include the pharmaceutically acceptable salts of any ofthe active substances. If active substances comprised by components (a)and/or (b) have, for example, at least one basic center, they can formacid addition salts. Corresponding acid addition salts can also beformed having, if desired, an additionally present basic center. Activesubstances having an acid group, e.g., COOH, can form salts with bases.The active substances comprised in components (a) and/or (b) or apharmaceutically acceptable salts thereof may also be used in form of ahydrate or include other solvents used for crystallization.

In one aspect, the composition comprises a second RNAi agent to EPAS1.In various aspects, the second RNAi agent is physically distinct fromthe first, or the two are physically connected (e.g., linked orconjugated). In some aspects, the first and second RNAi agents arecombined, within the same composition (e.g., both in the same lipidnanoparticle).

A Method of Inhibiting the Expression of EPAS1, Using an RNAi Comprisingan RNAi Agent Described Herein.

In one particular specific aspect, the present disclosure relates to amethod of inhibiting the expression of EPAS1 in an individual,comprising the step of administering to the individual a therapeuticallyeffective amount of a composition comprising an RNAi agent of thedisclosure. In one aspect, the RNAi comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense strand of an RNAi agent to EPAS1 selected from those specificduplexes provided above and as listed in Table 1.

In one aspect of the method, the RNAi agent comprises at least ananti-sense strand, and/or comprises a sense and an anti-sense strand,wherein the sequence of the sense and/or anti-sense strand is thesequence of the sense and/or the anti-sense strand of a RNAi agent toEPAS1 selected from those specific duplex provided herein and as listed,e.g., in Table 1, wherein the composition is in a pharmaceuticallyeffective formulation.

In one aspect of the method, the RNAi agent comprises at least ananti-sense strand, and/or comprises a sense and an anti-sense strand,wherein the sequence of the sense and/or anti-sense strand comprises thesequence of the sense and/or the anti-sense strand of a RNAi agent toEPAS1 selected from those specific duplex provided herein and as listed,e.g., in Table 1, wherein the composition is in a pharmaceuticallyeffective formulation.

Various particular specific aspects of this aspect are described below.

In one aspect, the individual is afflicted with or susceptible to anEPAS1-related disease.

In one aspect, the EPAS1-related disease is cancer, metastases,astrocytoma, bladder cancer, breast cancer, chondrosarcoma, colorectalcarcinoma, gastric carcinoma, glioblastoma, head and neck squamous cellcarcinoma, hepatocellular carcinoma, lung adenocarcinoma, neuroblastoma,non-small cell lung cancer, melanoma, multiple myeloma, ovarian cancer,rectal cancer, renal cancer, clear cell renal cell carcinoma (andmetastases of this and other cancers), gingivitis, psoriasis, Kaposi'ssarcoma-associated herpesvirus, preeclampsia, inflammation, chronicinflammation, neovascular diseases, and rheumatoid arthritis.

In one aspect, the EPAS1-related disease is cancer.

In one aspect, the method further comprises the step of administering anadditional treatment.

In one aspect, the additional treatment and the RNAi agent can beadministered in any order or can be administered simultaneously.

In one aspect, the method further comprises the step of administering anadditional treatment for cancer, metastases, astrocytoma, bladdercancer, breast cancer, chondrosarcoma, colorectal carcinoma, gastriccarcinoma, glioblastoma, head and neck squamous cell carcinoma,hepatocellular carcinoma, lung adenocarcinoma, neuroblastoma, non-smallcell lung cancer, melanoma, multiple myeloma, ovarian cancer, rectalcancer, renal cancer, clear cell renal cell carcinoma (and metastases ofthis and other cancers), gingivitis, psoriasis. Kaposi'ssarcoma-associated herpesvirus, preeclampsia, inflammation, chronicinflammation, neovascular diseases, and rheumatoid arthritis.

In one aspect, the composition comprises a second RNAi agent to EPAS1.In various aspects, the second RNAi agent is physically separate fromthe first, or the two are physically connected (e.g., covalently linkedor otherwise conjugated). In some aspects, the first and second RNAiagents are combined within the same composition (e.g., both in the samelipid nanoparticle).

In one aspect, the method further comprises the step of administering anadditional RNAi agent which comprises at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nucleotides from the antisense strand of aRNAi agent to EPAS1 selected from the specific duplexes provided hereinand as listed, e.g., in any Table herein.

Pharmaceutical Compositions of a RNAi Agent to EPAS1

In one particular specific aspect, the present disclosure relates to acomposition comprising a RNAi agent of the present disclosure. In oneaspect, the RNAi agent comprises at least an anti-sense strand, and/orcomprises a sense and an anti-sense strand, wherein the anti-sensestrand comprises at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nucleotides from the anti-sense strand of a RNAi agent to EPAS1selected from those specific duplex provided herein and as listed, e.g.,in Table 1, wherein the composition is in a pharmaceutically effectiveformulation.

In one aspect, the RNAi agent comprises at least an anti sense strand,and/or comprises a sense and an anti sense strand, wherein the sequenceof the sense and/or anti-sense strand is the sequence of the senseand/or the anti-sense strand of a RNAi agent to EPAS1 selected fromthose specific duplex provided herein and as listed, e.g., in Table 1,wherein the composition is in a pharmaceutically effective formulation.

In one aspect, the RNAi agent comprises at least an anti sense strand,and/or comprises a sense and an anti-sense strand, wherein the sequenceof the sense and/or anti-sense strand comprises the sequence of thesense and/or the anti sense strand of a RNAi agent to EPAS1 selectedfrom those specific duplex provided herein and as listed, e.g., in Table1, wherein the composition is in a pharmaceutically effectiveformulation.

In one aspect, the present disclosure pertains to the use of a RNAiagent in the manufacture of a medicament for treatment of aEPAS1-related disease, wherein the RNAi agent comprises a sense strandand an antisense strand, wherein the antisense strand comprises at least15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides fromthe antisense strand of a RNAi agent to EPAS1 selected from thosespecific duplex provided herein and as listed, e.g., any Table herein.

Specific Aspects of RNAi Agents to EPAS1 Comprising Mismatches from theDisclosed Sequences

Various specific aspects of a RNAi agent to EPAS1 are disclosed herein.The present disclosure encompasses the example modified variantsprovided in Tables 1 to 5 and the corresponding unmodified sequences andother modified variants. Specific aspects of the present disclosureinclude RNAi agents which comprise sequences differing by 0, 1, 2, or 3nt; (nucleotides) or bp [basepair(s)] (e.g., with 0, 1, 2 or 3mismatches) from any of the RNAi agents listed in Table 1, and modifiedand unmodified variants thereof. As described in additional detailbelow, a mismatch is defined herein as a difference between the basesequence (e.g., A instead of G) or length when two sequences aremaximally aligned and compared. In addition, as described in more detailbelow, an “unmodified variant” is a variant in which the base sequenceis identical, but none of the bases are modified; this includes, forexample, a sequence identical to the corresponding portion of thewild-type EPAS1 mRNA or gene. A “modified variant” contains one or moremodifications (or one or more fewer or different modifications) to anucleotide, sugar, phosphate or backbone, and/or addition of one or moremoieties; but without a change, substitution, addition, or deletion tothe base sequence. A particular sequence and its modified or unmodifiedvariants have 0 mismatches among them.

In one particular aspect, the present disclosure comprise RNAi agentcomprising a sense and an anti-sense strand, wherein the sense and/oranti sense strand comprises at least 15 contiguous nucleotides differingby 0, 1, or 3 nt from the sequence of the sense and/or anti-sense strandof: any of the RNAi age listed in Tables 1 to 5, and modified and unitunmodified variants thereof.

In another particular aspect, the RNAi agent comprises a sense strandcomprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3at form the sense strand of any of the RNAi agents listed in Tables 1and modified and unmodified variants thereof.

Other Aspects

Various particular specific aspects of this disclosure are describedbelow. Any aspects disclosed herein that are not mutually exclusive canbe combined.

In one aspect, the disclosure pertains to a composition according to anyof the above aspects, for use in a method of treating a EPAS1-relateddisease in an individual, the method comprising the step ofadministering to the individual a therapeutically effective amount of acomposition according to any of the claims.

Various particular specific aspects of this aspect are described below.

In one aspect, the disclosure pertains to the composition according toany of the above aspects, for use in a method of inhibiting theexpression of EPAS1 in an individual, the method comprising the step ofadministering to the individual a therapeutically effective amount of acomposition according to any of the above aspects.

One aspect of the disclosure is the use of a composition according toany of the above aspects, in the manufacture of a medicament fortreatment of an EPAS1-related disease.

In one aspect, the EPAS1-related disease is selected from cancer,metastases, astrocytoma, bladder cancer, breast cancer, chondrosarcoma,colorectal carcinoma, gastric carcinoma, glioblastoma, head and necksquamous cell carcinoma, hepatocellular carcinoma, lung adenocarcinoma,neuroblastoma, non-small cell lung cancer, melanoma, multiple myeloma,ovarian cancer, rectal cancer, renal cancer, clear cell renal cellcarcinoma (and metastases of this and other cancers), gingivitis,psoriasis, Kaposi's sarcoma-associated herpesvirus, preeclampsia,inflammation, chronic inflammation, neovascular diseases, and rheumatoidarthritis.

In one aspect, the disclosure pertains to the composition of any of theabove aspects, for use in the treatment of an EPAS1-related disease.

In one aspect, the EPAS1-related disease is cancer.

In one aspect, the disclosure relates to a method of inhibiting theexpression of EPAS1 in an cell, comprising the step of introducing intothe cell a composition comprising an RNAi agent comprising an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of an RNAi agent to EPAS1 selected from the EPAS1 siRNAsdisclosed herein.

In one aspect, the disclosure relates to a method of inhibiting theexpression of EPAS1 in an cell, comprising the step of introducing intothe cell a composition comprising an RNAi agent comprising a sensestrand and an antisense strand, wherein the antisense strand comprisesat least 15 contiguous nucleotides differing by 0, 1, 2, or 3nucleotides from the antisense strand, and the sense strand comprises atleast 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotidesfrom the sense strand of an RNAi agent to EPAS1 selected from the EPAS1siRNAs disclosed herein.

Definitions

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below. Ifthere is an apparent discrepancy between the usage of a term in otherparts of this specification and its definition provided in this section,the definition in this section shall prevail.

EPAS1

By “EPAS1” is meant the gene or protein also known as endothelial PASdomain protein 1, also known as EPAS-1, HIF-2 alpha; Hif2 alpha; HIF2;HLF; MOP2; ECYT4; HIF2A; PASD2; bHLHe73; HGNC: 3374; Gene ID: 2034. Notethat some references label the gene HIF-2a, and the correspondingprotein EPAS1 (for example, van Patot et al. 2011 High Alt. Med. Biol.12: 157-167); other documents use the same term to refer to both thegene and protein. See: Ema et al, 1997 Proc. Natl. Acad. Sci. USA 94:4273-4278; Flamme et al. 1997 Mech. Dev. 63: 51-60; and Hogenesch et al,1997 J. Biol. Chem. 272: 8581-8593. Various polymorphisms of EPAS1 areknown, as described in the literature, for example; van Patot et, al.2011 High Alt. Med. Biol. 12: 157-67; Ke et al. Zhonghua Yi Xue Yi ChuanXue Za Zhi. 2011 October; 28(5):583-8; PMID: 21983741.

This gene encodes a transcription factor involved in the induction ofgenes regulated by oxygen, which is induced as oxygen levels fall.

EPAS1 is a member of the HIF family. Hypoxia-inducible factors (HIFs)are transcription factors that respond to changes in available oxygen inthe cellular environment, specifically, to decreases in oxygen, orhypoxia. Smith et al. 2008 Br. J. Haematol, 141: 325-34.

Most, if not all, oxygen-breathing species express the highly-conservedtranscriptional complex HIF-1, which is a heterodimer composed of analpha and a beta subunit, the latter being a constitutively-expressedaryl hydrocarbon receptor nuclear translocator (ARNT), Wang et al. 1995Proc. Natl. Acad. Sci. USA 92: 5510-14; Jiang et al, 1996 J. Biol. Chem.271: 17771-8.

HIF family members include both Hif1 alpha and EPAS1 (Hif 2 alpha), thetwo best characterized HIF alpha subunits. While these two genes arehighly similar and bind and mediate many of the same targets, they aredifferent in function both temporally and spatially. While HIF1 alphadiminished the expression of interleukin-8 (IL-8), overexpression ofEPAS1 increases expression of IL-8 (Florczyk et al, 2011 Free Radic.Biol. Med. 51: 1882-92).

EPAS1 (Hif2 alpha) encodes a half of a transcription factor involved inthe induction of genes regulated by oxygen, which is induced as oxygenlevels fall (hypoxia). The encoded protein contains a basichelix-loop-helix domain protein dimerization domain as well as a domainfound in proteins in signal transduction pathways which respond tooxygen levels. EPAS1 is involved in the development of the embryonicheart and is expressed in the endothelial cells that line the walls ofthe blood vessels in the umbilical cord. It is essential in maintainingcatecholamine homeostasis and protection against heart failure duringearly embryonic development. Catecholamines include proteins such asepinephrine and norepinephrine. It is important for the production ofcatecholamines to remain in homeostatic conditions so that both thedelicate fetal heart and the adult heart do not overexert themselves andinduce heart failure. Catecholamine production in the embryo is relatedto control cardiac output by increasing the fetal heart rate.

Mutations in this gene are associated with erythrocytosis familial type4, pulmonary hypertension and chronic mountain sickness. There is alsoevidence that certain variants of this gene provide protection forpeople living at high altitude. EPAS1 is useful in high altitudes as ashort term adaptive response. However, EPAS1 can also cause excessiveproduction of red blood cells leading to chronic mountain sickness thatcan lead to death and inhibited reproductive abilities. Some mutationsthat increase its expression am associated with increased hypertensionand stroke at low altitude, with symptoms similar to mountain sickness.People permanently living at high altitudes might experience selectionof EPAS1 to reduce the fitness consequences of excessive red blood cellproduction.

EPAS1-Related Diseases

siRNAs to EPAS1 can be used to treat EPAS1-related diseases. An“EPAS1-related disease” is any disease associated with EPAS1 and/or amutation and/or an over-expression of a wild-type and/or mutant EPAS1,and/or diseases wherein disease progression is enhanced by or prognosisworsened by the presence of EPAS1 and/or a mutation and/or anover-expression of wild-type and/or mutant EPAS1. Non-limiting examplesof EPAS1-related diseases include: cancer, metastases, astrocytoma,bladder cancer, breast cancer, chondrosarcoma, colorectal carcinoma,gastric carcinoma, glioblastoma, head and neck squamous cell carcinoma,hepatocellular carcinoma, lung adenocarcinoma, neuroblastoma, non-smallcell lung cancer, melanoma, multiple myeloma, ovarian cancer, rectalcancer, renal cancer, clear cell renal cell carcinoma (and metastases ofthis and other cancers), gingivitis, psoriasis, Kaposi'ssarcoma-associated herpesvirus, preeclampsia, inflammation, chronicinflammation, neovascular diseases, and rheumatoid arthritis. Sec:Bangoura et al. 2004 World J. Gastroenterol. 10: 525; Cleven et al. 2007Analyt. Cell. Path. 29: 229-240; Covello et al. 2006 Genes Dev. 20:557-570; Florczyk et al. 2011 Free Radic. Biol. Med. 51: 1882-92;Giatromanolaki et al. 2003 Melanoma Res. 13: 493-501; Giatromanolaki etal. 2006 App. Imm. Mol. Morph. 14: 78-82; Giatromanolaki et al. 2012Clin. Exp. Metastasis 29: 11-7; Griffiths et al. 2008 Br. J. Cancer 98:965-973; Guo et al. J. Kanazawa Med. U. 31: 10-16; Holmquist-Mengelbieret al. 2006 Cancer Cell 10: 413-23; loachim et al. 2006 Urol. Int. 77:255-263; Koh et al. 2011 cancer Res. 71: 4015-4027; Maynard et al. 2007Cell. Mol. Life Sci. 64: 2170-2180; Mutter et al. 2008 Microvasc. Res.75: 1-8; Nesbit et al. 1999 Oncogene Mol. Cell 3: 565-577; Osada et al.2007 Human Pathol. 38: 1310-1320: Pelegaris et al. 2002 Nat. Re. Cancer2: 764-776; Rasheed et al. 2009 Br. J. Cancer 100: 1666-1673; Tian etal. 1998 Genes Dev. 12:3320-3324; Vecranna et al. 2012 J. Virol. 86:1097-708; Xu et al. 2012 Oncogene 31: 1065-72:

HIF induction in normoxia is likely to have serious consequences indisease settings with a chronic inflammatory component. It has also beenshown that chronic inflammation is self-perpetuating and that itdistorts the microenvironment as a result of aberrantly activetranscription factors. Consequently, alterations in growth factor,chemokine, cytokine and ROS balance occur within the cellular milieuthat in turn provide the axis of growth and survival needed for de novodevelopment of cancer and metastasis. The results of a recentlypublished study have numerous implications for a number of pathologieswhere NF-κB and HIF-1 are deregulated, including rheumatoid arthritisand cancer. Therefore, it is thought that understanding the cross talkbetween these two key transcription factors, NF-κB and HIF, will greatlyenhance the process of drug development.

HIF activity is involved in angiogensis required for cancer tumorgrowth, so HIF inhibitors such as phenethyl isothiocyanate (PEITC) canbe used for anti-cancer effects. At least part of the role of EPAS1 intumor progression has been assigned to EPAS1-mediated upregulation ofvarious genes. For example, EPAS1 plays an important role in theprogression of uveal melanomas, possibly by promoting the autocrine loopVEGF-pVEGFR2/KDR, and by enhancing the expression of LDHA, thusconferring a growth advantage; see Giatromanolaki et al. 2012 Clin. Exp.Metastasis 29: 11-7.

EPAS1 is also involved in or upregulates expression of these factors:c-Myc (which favors cell proliferation, transformation, neoplasia andtumorigenesis, and which is highly expressed in most cancers; seePelegaris et al. 2002 Nat. Re. Cancer 2: 764-776; Nesbit et al. 1999Oncogene Mol. Cell 3: 565-577; Florczyk et al. 2011 Free Radic. Biol.Med. 51: 1882-92); Interleukin 8 (a proinflammatory mediator, e.g., ingingivitis and psoriasis; see Florczyk et al. 2011 Free Radic. Biol.Med. 51: 1882-92); SP-1 (a transcription factor involved in IL-8regulation and a coactivator of c-Myc; see Florczyk et al. 2011 FreeRadic. Biol. Med. 51: 1882-92); LDH5 (which is linked with tumornecrosis and increased tumor size; see Giatromanolaki et al. 2012 Clin.Exp. Metastasis 29: 11-7); and LANA (Latency Associated Nuclear Antigen,which is associated with Kaposi's sarcoma-associated Herpesvirus; seeVeeranna et al. 2012 J. Virol. 86: 1097-708). In addition tocollaborating with c-Myc, EPAS1 also collaborates with EGFR and KRAS;see, Holmquist-Mengelbier et al. 2006 Cancer Cell 10: 413-23; and Koh etal. 2011 Cancer Res. 71: 4015-4027. Thus, any disease related toover-expression and/or hyperactivity of c-Myc, EGFR and KRAS can beconsidered to be a EPAS1-related disease. In addition, HIF (hypoxiainduced factor) activity is involved in angiogenxis required for cancertumor growth.

There is a rationale for targeting clear cell renal cell carcinoma (RCC)(ccRCC) and metastases thereof with EPAS1 RNAi agents. First, 90% ofccRCC cells do not express the VHL tumor suppressor. Second, in absenceof VHL tumor suppressor Hif transcription factors are constitutivelyactivated. Third, expression of Hif-2 is necessary and sufficient forRCC xenograft growth according to published studies. Constitutive Hif-2shRNA knockdown blocks growth of 786-O cell xenografts. Inducible Hif-2shRNA knockdown demonstrates that Hif-2 is necessary for 786-O xenograftmaintenance. Sec also: Tian et al. 1998 Genes Dev. 12:3320-3324;Veeranna et al. 2012 J. Virol. 86: 1097-708; and Xu et al. 2012 Oncogene31: 1065-72; Zimmer et al. 2004. Molecular Cancer Research 2:89-95;Kondo et al. 2002. Cancer Cell 1:237-246; and Kondo et al. 2003. PLoSBiology 1:439-444.

Thus, the present disclosure encompasses EPAS1 RNAi agents and the usesthereof for EPAS1-related diseases.

EPAS1 Gene Sequences in Various Species

The human EPAS1 gene has been sequenced. Ema et al. 1997 Proc. Natl.Acad. Sci. USA 94: 4273-4278; Flamme et al. 1997 Mech. Dev. 63: 51-60;and Hogenesch et al. 1997 J. Biol. Chem. 272: 8581-8593. Variouspolymorphisms of EPAS1 are known, as described in the literature, forexample: van Patot et al. 2011 High Alt. Med. Biol. 12: 157-67; Ke etal. Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2011 October; 28(5): 583-8;PMID: 21983741.

The Cynomolgus monkey (“Cyno”, Macaca fascicularis) EPAS1 sequence (SEQID NO: 125) is presented below:

cyno_kidney_NM_001430 EPAS1, endothelial PAS domain protein 1 consensus -- target length =5186, consensus length = 4636, 9 contigs (longest is 2117) 104 mismatches, 1352 reads, coverage: max = 84, mean = 18.499; consensus consistency = 99.31%, target/consensus conservation 97.76% (SEQ ID NO: 125)NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCTCGGCAGTcTCCTGAGACTGTATGGTCAGCTCAGCCCAGCCTCCGACTCCTTCCGACTCCCAGCATTCGAGCCACTTTTTTTTTTCCTTGAAAACTCAGAAAAGTGACTCTTTTTCCAGGGAAAAAGGAACTTGGGTTCCCTTCTCGCCGTCCTTTTTTCGGGTCTGACAGCCTCCACCCACTCCTTCCCCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCGTCACCTTTCTCCACCCCCACCCCCGCACCTAGCCCGCCGCGCGCCACCTTCCACCTGACTGCGCGGGGCGCTCGGGACCTGCGCGCACCTCGGACCTTCACCACCCGCCCcGGCCGCCGGGAGCGGACGAGGGCCACAGCTCCCCACCCGCCGGGAAGCCCAGGTGCTCGGCGTCTGAACGTCTCAAAGGGCCACAGCGACAATGACAGCTGACAAGGAGAAGAAAAGGAGTAGCTCGGAGAGGAGGAAGGAGAAGTCCCGGGATGCCGCACGGTGCCGGCGGAGCAAGGAGACGGAGGTGTTCTACGAGCTGGCCCATGAGCTGCCTCTGCCCCACAGCGTGAGCTCCCATCTGGACAAGGCCTCCATCATGCGACTGGCGATCAGCTTCCTGCGAACACACAAGCTCCTCTCCTCAGTTTGCTCTGAAAATGAGTCTGAAGCTGAAGCTGACCAGCAGATGGACAACTTGTACCTGAAAGCCTTGGAGGGTTTCATTGCCGTGGTGACCCAAGATGGCGACATGATCTTTCTGTCAGAAAACATCAGCAAGTTCATGGGACTTACACAGGTGGAGCTAACAGGACATAGTATCTTTGACTTCACTCATCCCTGCGACCACGAGGAGATTCGTGAGAACCTGAGTCTCAAAAATGGCTCTGGTTTTGGGAAAAAAAGCAAAGACATGTCCACAGAGCGGGACTTCTTCATGAGGATGAAGTGCACGGTCACCAACAGAGGCCGTACTGTCAACCTCAAGTCAGCCACCTGGAAGGTCTTGCACTGCACGGGCCAAGTGAAAGTCTACAACAACTGCCCTCCTCACAATAGTCTGTGTGGCTACAAGGAGCCCCTGCTGTCCTGCCTCATCATCATGTGTGAACCGATCCAGCACCCATCCCACATGGACATTCCCCTGGACAGCAAGACCTTCCTGAGCCGCCACAGCATGGACATGAAGTTCACCTACTGTGATGACAGAATCACAGAACTGATTGGTTACCACCCTGAGGAGCTGCTTGGCCGCTCAGCCTATGAATTCTACCATGCGCTAGACTCCGAGAACATGACCAAGAGTCACCAGAACTTGTGCACCAAGGGCCAGGTGGTAAGTGGCCAGTACCGGATGCTCGCAAAGCATGGGGGCTACGTGTGGCTGGAAACCCAGGGGACAGTCATCTACAACCCTCGCAACCTGCAGCCCCAGTGCATCATGTGTGTCAACTACGTTCTGAGTGAGATTGAGAAGAATGACGTGGTGTTCTCCATGGACCAGACGGAATCCCTGTTCAAGCCCCACCTGATGGCCATGAACGGCATCTTTGATAGCAGTGGCAAGGGGGCTGTGTCTGAGAAGAGTAACTTCCTATTCACCAAGCTAAAGGAGGAGCCTGAGGAGCTGGCCCAGCTGGCTCCCACCCCAGGAGACGCCATCATCTCTCTGGATTTCGGGAATCAGAACTTCGAGGAATCCTCAGCCTATGGCAAGGCCATCCTGCCCCCGAGCCAGCCGTGGGCCACAGAGTTGAGGAGCCACAGCACCCAGAGCGAGGCTGGGAGCCTGCCTGCCTTCACCGTGCCCCAGGCAGCCGCCCCGGGCAGCACCACCCCCAGTGCCACCAGCAGCAGCAGCAGCTGCTCCACGCCCAATAGCCCTGAAGACTATTATACATCTTTGGATAACGACCTGAAGATTGAAGTGATTGAGAAGCTCTTCGCCATGGACACAGAGGCCAAGGACCAATGCAGTACCCAGACGGATTTCAATGAGCTGGACTTGGAGACACTGGCACCCTATATTCCCATGGATGGGGAAGACTTCCAGCTGAGCCCCATCTGCCCCGAGGAGCGGCTCTTGGCGGAGAACCCACAGTCCACCCCCCAGCACTGCTTCAGTGCCATGACAAACATCTTCCAGCCACTGGCCCCTGTAGCCCCGCACAGTCCCTTCCTCCTGGACAAGTTTCAGCAGCAGCTGGAGAGCAAGAAGACAGAGCCCGAGCACCGGCCCATGTCCTCCATCTTCTTTGATGCCGGAAGCAAAGCATCCCTGCCACCATGCTGTGGCCAGGCCAGCACCCCTCTCTCTTCCATGGGGGGCAGATCCAATACCCANTGGCCCCCAGATCCACCATTACATTTTGGGCCCACAAAGTGGGCCGTCGGGGATCAGCGCACAGAGTTCCTGGGAGCGNNNNNNNNNNNNNNNNNNNNNNNNNNNNCCCATATCTCCACATTCAAGACAAGGTCTGCAAAGGGTTTTGGGGCTCGAGGCCCAGACGTGCTGAGCCCGGCCATGGTAGCCCTCTCCAACAAGCTGAAGCTGAAGCGACAGCTGGAGTATGAAGAGCAAGCCTTCCAGGACCTGAGTGGGGGGGACCCACCTGGTGGCAGCACTTCACATTTGATGTGGAAACGGATGAAGAACCTCAGGGGTGGGAGCTGCCCTTTGATGCCGGACAAGCCACTGAGCGCAAATGTCCCCAATGGTAAGTTCACCCAAAATCCTGTGAGGGGCCTGGGCCATCCCCTGAGACATCTGCCGCTGCCACAGCCTCCATCTGCCGTCAGTCCCGGGGAGAACAGCAAGAGCAGGTTCCCCGCACAGTGCTATGCCACcCAGTACCAGGACTACAGCCTGTCGTCAGCCCACAAGGTGTCAGGCATGGCAAGCCGGCTGCTCGGGCCCTCGTTTGAGTCCTACCTGCTGCCTGAACTGACCAGATATGACTGTGAGGTGAACGTGCCCGTGCTGGGAAGCTCCACGCTCCTGCAAGGAGGGGACCTCCTCAGAGCCCTGGACCAGGCCACCTGAGCCAGGCCTTCCACCTGGGCAGCACCTCTGCCGACACCGTCCCACCAGCTTCACTCTCTCCATCTGTTTTTGTAACTAGGTATTTCTAACACCAGCACACTATTTACAAGATGGACTTACCTGGCAGACTTGCCCAGGTCACCACGCAGTGGCCTTTTTCTGAGATGCTCACTTTATTATCCCTATTTTTAAAGTACACAATTGTTTTACCTGTTCTGAAATGTTCTTAAATTTTGTAATATTTTTTTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGCGTTAGCTTCATTTTACTAAAAAGATTCCTCGTTACTGTTGTTGCCAAAGAGAAACAAAAATGATGTTGCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAAAAAAGAAATGTGAAGGGTCAACTCCAACGTATGTGGTTATCTGTGAAGGCTGCATAGCGTGGCTTTTCCTAAACTGGTGTTTTTCCCCCGCATTCGGTGGATTTTTTATTATTATTCAAAAACATAACTGAGTTTTTNNNNNNNNNAGAAAATTTATATCTGGGTTAAGTGTTTATCATATATATGGGTACTCTGTAATATCTAAAACCTTAGAAACGGAAATGGAATCCTGCTCACAAAATCACTTTAAGATCTTTTCAAAGCTGTTAATTTTTCTTGGTGTTGTGGACACTGCAGACTTGTCCAGTGCTCCCACAGCCTGTACGGACACTGTGGAAGGCCTCCCTCTGTCGGCTTTTTGCCATCTGTGATATGCCATAGGTGTGACAATCCGAGCAGTGGAGTCATTCAGTGGGAGCACTGCGCGCTATCCCCTCATGTTCTCTATGTACTATGTATGTATGTATTATTATTATTGCTGCCAAGAGGGTCTGATGGCACGTTGTGGGGTCGGGGGGTGGGGCGGGGAAGTGCTCTAACTTTTCTTAAGGTTTTGTTGCTAGCCCTTCAAGTGCACTGAGCTATGTGACTCGGATGGTCTTTCACACGGCACATTTAGACATTTCCAGAACTACCATGAGATGGTTTAGATGGGAATTCATGCAAATGAGGGGTCAGAAATGGTATAGTGACCCGGTCCACGTCCTCCAAGCTCACGACCTTGGAGCCCCGTGGAGCTGGACTGAGGAGGAGGCTGCACAGCGGGAGAGCAGCTGGTCCAGACCAGCCTTGCAGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAAGCACTGAAAATAGCGTTCCCAGAGCACATTGCAACTCACTGGGTAAGAGGGACGACACCTCTGGTTTTTCAATACCAATTACATGGAACTTTTCTGTAATGGGTACAACGAAGAAGTTTCTAAAAACACACACAAAGCACATTAGGCCAACTATTTAGTAAGCCCGGATGGACTTATTGCCAGAAACAAAAAGTAGCTTTCAAAAGAAATTTAAGTTATATGAGAAATTCCTTAGTCATGGTGTTGTCTAAATCATATTTTAGCTGCACGNNNNNNNNNNNNNNNNNNNGGCAGAACTTGAAGGGTTACTGACATGTAAATGCTGGTATTTGATTTCCTGTGTGTGTTGCCCTGGCATTAAGGGCATTTTACCCTTGCAGTTTTACTAAAACACTGAAAAATATTCCAAGCTTCATATTAACCCTACCTGTCAACGTAACGATTTCATGAACATTATTATATTGTCGAATTCCTACTGACAACATTATAACTGTATGGGAGCTTAACTTTATAAGGAAATGTATTTTGACACTGGTATCTTATTAAAGTATTCTGATCCTAAAANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

N indicates that the nucleotide was not determined at that position inthe sequencing experiment.

In one aspect, the EPAS1 RNAi agent of the present disclosure comprisesa sequence which is identical in the human, mouse and cyno EPAS1 gene.This sequence identity facilitates animal testing prior to humantesting.

In one aspect, the EPAS1 RNAi agent comprises a sequence which does notmatch that of any other gene. In one aspect, the EPAS1 RNAi agentcomprises a sequence which differs from all other known non-EPAS1 genesby at least 0, 1, 2 or 3 nucleotides.

In one aspect, the EPAS1 RNAi agent comprises a sequence which isidentical to that of any RNAi agent disclosed herein.

EPAS1 RNAi Agent for Use in Treating Various EPAS1-Related Diseases

In one aspect, the EPAS1 RNAi agent of the present disclosure comprisesa sequence disclosed herein and is administered to a patient in needthereof (e.g., a patient suffering from an EPAS1-related diseasedisclosed herein or known in the literature). In one aspect, the EPAS1RNAi agent of the present disclosure is administered to a patient inneed thereof, along with one or more additional pharmaceutical agentappropriate for that disease. For example, a patient suffering from anEPAS1-related disease can be administered a pharmacologically effectiveamount of one or more EPAS1 RNAi agent along with a pharmacologicallyeffective amount of one or more of any EPAS1-related disease treatmentlisted herein, and/or any other EPAS1-related disease treatment known inthe art.

A patient suffering from a EPAS1-related disease can be administered oneor more RNAi agent to EPAS1 and one or more additional EPAS1-relateddisease treatment. This additional treatment can be selected from thelist of any disease treatment listed herein, and/or anyanti-EPAS1-related disease treatment known in the art.

The patient can also be administered more than one RNAi agent to EPAS1.

In the ease of EPAS1-related diseases, the RNAi agent(s) and additionaldisease treatments) can be administered in any order, simultaneously orsequentially, or in multiple doses over time. Administration of the RNAiagent and the additional treatment can be for example, simultaneous,concurrent, separate or sequential.

Simultaneous administration may e.g., fake place in the form of onefixed combination with two or more active ingredients, or bysimultaneously administering two or more active ingredients that areformulated independently. Sequential use (administration) preferablymeans administration of one (or more) components of a combination atone, time point, other components at a different time point, that is, ina chronically staggered manner, preferably such that the combinationshows more efficiency than the single compounds administeredindependently (especially showing synergism). Separate use(administration) preferably means administration of the components ofthe combination independently of each other at different time points,preferably meaning that the components (a) and (b) are administered suchthat no overlap of measurable blood levels of both compounds are presentin an overlapping manner (at the same time).

Also combinations of two or more of sequential, separate andsimultaneous administration are possible, preferably such that thecombination component-drugs show a joint therapeutic effect that exceedsthe effect found when the combination component-drugs are usedindependently at time intervals so large that no mutual effect on theirtherapeutic efficiency can be found, a synergistic effect beingespecially preferred.

The term “delay of progression” as used herein means administration ofthe combination to patients being in a pre-stage or in an early phase,of the first manifestation or a relapse of the disease to be treated, inwhich patients, e.g., a pre-form of the corresponding disease isdiagnosed or which patients are in a condition, e.g., during a medicaltreatment or a condition resulting from an accident, under which it islikely that a corresponding disease will develop.

“Jointly therapeutically active” or “joint therapeutic effect” meansthat the compounds may be given separately (in a chronically staggeredmanner, especially a sequence-specific manner) in such time intervalsthat they preferably, in the warm-blooded animal, especially human, tobe treated, still show a (preferably synergistic) interaction (jointtherapeutic effect). Whether this is the case, can inter alia bedetermined by following the blood levels, showing that both compoundsare present in the blood of the human to be treated at least duringcertain time intervals.

Additional Definitions

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below. Ifthere is an apparent discrepancy between the usage of a term in otherparts of this specification and its definition provided in this section,the definition, in this section shall prevail.

As used throughout this disclosure, articles such as “a” and “an” referto one or more than one (at least one) of the grammatical object of thearticle.

RNAi Agent

In one aspect, the present disclosure pertains to a EPAS1 RNAi agent orother composition comprising at least an antisense nucleic acid sequencecomplementary to a EPAS1 nucleic acid (or portion thereof), or pertainsto a recombinant expression vector encoding an shRNA or compositioncomprising the antisense nucleic acid That can function as an RNAi asdefined below. As used herein, an “antisense” nucleic acid comprises anucleotide sequence complementary to a “sense” nucleic acid encoding theEPAS1 protein (e.g., complementary to the coding strand of adouble-stranded DNA, complementary to an mRNA or complementary to thecoding strand of a EPAS1 gene or nucleic acid).

RNAi agents include, as non-limiting examples, siRNAs (small interferingRNAs), dsRNAs (double stranded RNAs), shRNAs (short hairpin RNAs) andmiRNAs (micro RNAs). RNAi agents also include, as additionalnon-limiting examples, looked nucleic acid (LNA), Morpholino, UNA,threose nucleic acid (TNA), or glycol nucleic acid (GNA), peptidenucleic acid (PNA) and FANA. RNAi agents also include molecules in whichone or more strands are a mixture of RNA, DNA, LNA, Morpholino, UNA(unlocked nucleic acid), TNA, GNA, and/or FANA, etc. As a non-limitingexample, one or both strands of an RNAi agent could be, for example,RNA, except that one or more RNA nucleotides is replaced by DNA, LNA,Morpholino UNA (unlocked nucleic acid), TNA, GNA, and/or FANA, etc. invarious aspects, one or both strands of the RNAi agent can be nicked,and both strands can be the same length, or one (e.g., the passengerstrand), can be shorter than the other.

In various aspects, the present disclosure pertains to any RNAi agentcomprising a RNA sequence disclosed herein and/or a RNA sequencecorresponding to any DNA sequence disclosed herein (e.g., wherein theDNA nucleotides are replaced by the corresponding RNA nucleotide, forexample, with T in DNA replaced by U in RNA, and with ribose instead ofdeoxyribose in the sugar-phosphate backbone).

The RNAi agent(s) of the present disclosure target (e.g., bind to,anneal to, hybridize, etc.) the EPAS1 mRNA. The use of the RNAi agentspecific to EPAS1 results in a decrease of EPAS1 activity, level and/orexpression, e.g., a “knock-down” (KD) or “knock-out” of the target geneor target sequence. In one aspect, in the case of a disease statecharacterized by over-expression or hyper-activity of EPAS1,administration of a RNAi agent to EPAS1 knocks down the EPAS1 targetenough to provide a more normal or therapeutic level of EPAS1 activityor expression. EPAS1 −/− mice showed multiple organ pathology,biochemical abnormalities and altered gene expression; see Scortegagnaet al. Nat. Genet. 35: 331-340. Thus, a minimal expression of EPAS1 innormal tissues can be beneficial. In various aspects of the disclosure,the patient or individual may have a disease state characterized byexcessively high levels of EPAS1 and the RNAi agent can restore a normallevel. In one aspect of the disclosure, the levels of EPAS1 throughoutthe body are modulated such that EPAS1 levels in one area (e.g., areasafflicted by a EPAS1-related disease) are lower, while areas of the bodynot afflicted by the disease are closer to normal EPAS1 levels. In oneaspect of the disclosure, the RNAi agent can be delivered locally (e.g.,to the site of the disease, such as a tumor) so that levels of EPAS1outside the diseased areas can be maintained as close to normal aspossible. In another aspect, the level of EPAS1 in the body can bemodulated such that it is low enough to improve the disease state (e.g.,low enough to discourage tumor growth), but not so low that organpathology occurs.

In one aspect, the RNAi comprises a single strand. This single-strandedRNAi agent oligonucleotide or polynucleotide can comprise the sense orantisense strand, as described by Sioud 2005 J. Mol. Biol.348:1079-1090, and references therein. Thus the disclosure encompassesRNAi agents with a single strand comprising either the sense orantisense strand of an RNAi agent described herein. The disclosure alsoencompasses RNAi agents comprising a single strand, wherein the singlestrand comprises the sequences of both the antisense and sense strandsof any RNAi agent disclosed herein, e.g., wherein the strands arecontiguous, connected by a loop or otherwise linked. Examples of suchmolecules include those with a hairpin between the sense and anti-sensesequences (e.g., shRNA).

In various aspects, one or both strands contain one or more nicks, i.e.,a break or missing bond in the phosphate backbone, such that at leastone nucleotide subunit is not covalently linked to the adjacentnucleotide subunit in any given sequence. In some aspects, the passengerstrand is nicked (see, for example, WO 2007/107162). In various aspects,one or both strands contain one or more gaps, e.g., wherein at least oneentire nucleotide subunit is absent from the disclosed sequence. Where asense or antisense sequence contains a gap, that strand is envisioned tocomprise two separate oligonucleotides.

Particularly useful siRNAs include those which can bind specifically tothose regions of the EPAS1 mRNA that have one or more of the followingqualities: binding in the coding segment of EPAS1; binding at or nearthe junction of the 5′ untranslated region and the start of the codingsegment; binding at or near the translational start site of the mRNA;binding at, across or near junctions of exons and introns; little or nobinding to the mRNAs or transcripts of other genes (little or no“off-target effects”); binding to the EPAS1 mRNA in or near a region orregions that is not double-stranded or a stem region, e.g., those in aloop or single-stranded portion; eliciting little or no immunogenicity;binding in a segment of the EPAS1 mRNA sequence which is conserved amongvarious animal species (including human, mouse, rat, cyno, etc.), as thepresence of a conserved sequence facilitates testing using variouslaboratory animals; binding to double-stranded region(s) of the mRNA;binding to an AT-rich region (e.g., at least about 50, 51, 52, 53, 54,55, 56, 57, 58, 59, or 60% AT-rich); and/or lacking particular sequencesknown or suspected to decrease siRNA activity, e.g., the presence of aGG sequence at the 5′ end, which may decrease separation of thedouble-stranded portion of the siRNA. In one aspect, the RNAi agentspecific to EPAS1 can be a double-stranded RNA having any one or more ofthese qualities.

The term “double-stranded RNA” or “dsRNA,” as used herein, refers to aRNAi agent comprising a first and a second strand; e.g., a compositionthat includes an RNA molecule or complex of molecules having ahybridized duplex region (i.e., a region where the nucleotide bases fromthe first strand and the second strand are paired) that comprises twoanti-parallel and substantially complementary nucleic acid strands,which will be referred to as having “sense” and “antisense” orientationswith respect to a target RNA. The antisense strand, with respect to themRNA target, is also called the “guide” strand, and the sense strand isalso called the “passenger” or “anti-guide” strand. The passenger strandcan include at least one or more of the following: one or more extranucleotides (e.g., a bulge or 1 nt loop) compared to the other strand,and/or a nick, a gap, a mismatch, etc., compared to the other strand. Invarious aspects, the RNAi agent comprises a first strand and a secondstrand. In various aspects, and as used herein, and as is clear bycontext, terminology referring to the first strand refers to the sensestrand and the second strand refers to the anti sense strand as listedin any Table herein. In other aspects, and as used herein and as isclear by context, the first strand refers to the anti-sense strand, andthe second strand refers to the sense strand as listed in any Tableherein.

The duplex region can be of any length that permits specific degradationof a desired target RNA through a RISC pathway, but will typically rangefrom 9 to 36 base pairs (“bp”) in length, e.g., 15-30 bp in length.Considering a duplex between 9 and 36 bp, the duplex can be any lengthin this range, for example, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 bpand any sub-range therebetween, including, but not limited to 15-30 bp,15-26 bp, 15-23 bp, 15-22 bp, 15-21 bp, 15-20 bp, 15-19 bp, 15-18 bp,15-17 bp, 18-30 bp, 18-26 bp, 18-23 bp, 18-22 bp, 18-21 bp, 18-20 bp,19-30 bp, 19-26 bp, 19-23 bp, 19-22 bp, 19-21 bp 19-20 bp, 19 bp, 20-30bp, 20-26 bp, 20-25 bp, 20-24 bp, 20-23 bp, 20-22 bp, 20-21 bp, 20basepairs, 21-30 bp, 21-26 bp, 21-25 bp, 21-24 bp, 21-23 bp, 21-22 bp,21 bp, 22 bp, or 23 bp. The dsRNAs generated in the cell by processingwith Dicer and similar enzymes are generally in the range of about 19 toabout 22 bp in length, though the strands of artificial dsRNAs can beshorter or longer. siRNAs wherein one or both strands are as short as 16or 15 nt still demonstrate RNA interference activity, Chu and Rana 2008RNA 1714-1719, One strand of the duplex region of a dsRNA comprises asequence that is substantially complementary to a region of a targetRNA. The two strands forming the duplex structure can be from a singleRNA molecule having at least one self-complementary duplex region, orcan be formed from two or more separate RNA molecules that hybridize toform the duplex. Where the duplex region is formed from twoself-complementary regions of a single molecule, the molecule can have aduplex region separated by a single-stranded chain of nucleotides(herein referred to as a “hairpin loop”, e.g., such as found in an shRNAconstruct) between the 3′-end of one strand and the 5′-end of therespective other strand forming the duplex structure. The hairpin loopcan comprise at least one unpaired nucleotide; in some aspects thehairpin loop can comprise at least 3, at least 4, at least 5, at least6, at least 7, at least 8, at least 9, at least 10, at least 20, atleast 23 or more unpaired nucleotides. Where the two substantiallycomplementary strands of a dsRNA are comprised by separate RNAmolecules, those molecules need not, but can, be covalently connected.Where the two strands are connected covalently by a hairpin loop, theconstruct is generally referred to herein and in the art as a “shRNA”.Where the two strands are connected covalently by means other than ahairpin loop, the connecting structure is referred to as a “linker.”

RNAi Agents to EPAS1 Comprising Mismatches from the Disclosed Sequences

Various specific aspects of a RNAi agent to EPAS1 are disclosed herein;example sequences are provided in the Tables. Specific aspects of thepresent disclosure include RNAi agents which comprise sequencesdiffering by 0, 1, 2, or 3 nt (nucleotides) or bp [basepair(s)] (e.g.,with 0, 1, 2 or 3 mismatches) from any of the RNAi agents listed inTables 1 to 5, and modified and unmodified variants thereof.

A mismatch is defined herein as a difference between the base sequenceor length when two sequences are maximally aligned and compared. Amismatch is defined as a position wherein the base of one sequence doesnot match the base of the other sequence. Thus, a mismatch is counted,for example, if a position in one sequence has a particular base (e.g.,A), and the corresponding position on the other sequence has a differentbase (e.g., G). Substitution of A, for example, with T, C, G or U wouldconstitute a mismatch. Substitution of G with T, A, C or U would alsoconstitute a mismatch. Substitution of C with T, G, A or U would alsoconstitute a mismatch. Substitution of U with A, C or G would constitutea mismatch. Note, however, that on a given strand, a U can be replacedby T (either as RNA or, preferably, DNA, e.g., 2′-deoxy-thymidine); thereplacement of a U with a T is not a mismatch as used herein, as eitherU or T can pair with A on the opposite strand. The RNAi agent can thuscomprise one or more DNA bases, e.g., T. In some cases, in a portion orportions of the RNAi agent. DNA can be used in place of RNA (e.g., inthe seed region), to form a DNA-RNA hybrid. See, for example, Yamato etal. 2011 cancer Gene Ther. 18: 587-597. No mismatch is counted between aDNA portion(s) of the RNAi agent and the corresponding target mRNA ifbasepairing occurs (e.g., between A, G, C, or T in the DNA portion, andthe corresponding U, C, G, or A, respectively in the mRNA).

A mismatch is also counted, e.g., if a position in one sequence has abase (e.g., A), and the corresponding position on the other sequence hasno base (e.g., that position is an abasic nucleotide, which comprises aphosphate-sugar backbone but no base). A single-stranded nick in eithersequence (or in the sense or anti-sense strand) is not counted asmismatch. Thus, as a non-limiting example, no mismatch would be countedif one sequence comprises the sequence AG, but the other sequencecomprises the sequence AG with a single-stranded nick between the A andthe G. A nucleotide modification in the sugar or phosphate is also notconsidered a mismatch. Thus, if one sequence comprises a C, and theother sequence comprises a modified C (e.g., 2′-modification) at thesame position, no mismatch would be counted.

Thus, no mismatches are counted if modifications are made to the sugar,phosphate, or backbone of the RNAi agent without modifying the base.Thus, a strand having the sequence of AUGGCGACAUGAUCUUUCU (SEQ ID NO: 1)as an RNA would have zero mismatches from another strand having the samesequence as a PNA; or morpholino; or LNA; or TNA; or GNA; or FANA; or amix or chimera of RNA and DNA, TNA, GNA, FANA, Morpholino, UNA, LNA,and/or PNA, etc.

It is also noted that the sequences of the RNAi agents in the Tablesinclude sequences which comprise modifications, as detailed in Table 5.It is noted that dTdT (2′-deoxy-thymidine-5′-phosphate and2′-deoxy-thymidine-5′-phosphate), or in some cases, TT or UU, can beadded as a terminal dinucleotide cap or extension to one or both3′-ends, but this cap or extension is not included in the calculation ofthe total number of mismatches and is not considered part of the targetsequence. This is because the terminal dinucleotide protects the endsfrom nuclease degradation but does not contribute to target specificity(Elbashir et al. 2001 Nature 411: 494-498; Elbashir et al. 2001 EMBO J.20: 6877-6888; and Kraynack et al. 2006 RNA 12:163-176).

In addition, as in Table 5, a modified variant can have one or moremodifications from the corresponding unmodified sequence. In this case,lowercase “c” represents 2′-O-methylcytidine-5′-phosphate, and lowercase“u” represents 2′-O-methyluridine-5′-phosphate, Uppercase “A”, “C”, “G”and “U” represent the un-modified adenosine-5′-phosphate,cytidine-5′-phosphate, guanosine-5′-phosphate, and uridine-5′-phosphate,respectively. Various modifications are shown in FIG. 1. Thesubstitution, for example, r of modified c for unmodified C does notcount as a mismatch in numbering the 0, 1, 2, or 3 mismatches betweensequences. This nomenclature is used for all sequences in Tables 1 to 6.Thus, an equal number of mismatches would be calculated (a) between atest sequence and that of another RNAi agent, and (b) between the sametest sequence and the corresponding unmodified sequence from the EPAS1gene, and (c) between a modified sequence and a differently modifiedsequence which have the same base sequence.

In one particular aspect, the present disclosure comprises a RNAi agentcomprising a anti-sense strand comprising at least 15 to 19 contiguousnucleotides differing by 0, 1, 2, or 3 at from the sequence of theanti-sense strand of: any of the RNAi agents listed in Tables 1 to 6,and modified and unmodified variants thereof.

The present disclosure pertains to “modified and unmodified variants” ofthe disclosed sequences.

An “unmodified variant” of a particular sequence is the correspondingportion of EPAS1 without any modifications. Example modified sequencesare listed in Table 3. The “unmodified variants” of the sequences of theTables have the identical sequence, without base modifications orterminal dTdT. A given sequence and an “unmodified variant” of it differby 0 nt and have no mismatches).

A “modified variant” of a particular sequence comprises one or more orone or more fewer) modifications to the backbone, sugar, phosphate orbase, and/or addition of a terminal dinucleotide (e.g., TT, dTdT, TsT orUU), but do not have any base substitutions (e.g., G for C, or A for G);thus a given sequence and a modified variant thereof differ by 0 nt (andhave no mismatches). As another example, a given sequence as a RNA andthe same sequence as a PNA are modified variants of each other anddiffer by 0 nt (and have no mismatches). Similarly, the same sequence(with no base substitutions) as a locked nucleic acid (LNA), Morpholino,UNA (unlocked nucleic acid), threose nucleic acid (TNA), or glycolnucleic acid (GNA) would be a modified variant which has 0 mismatches.In addition, the same sequence could be used in strands which are amixture of RNA, DNA, LNA, Morpholino, TNA, GNA, and/or FANA, etc. As anon-limiting example, one or both strands could be, for example, RNAexcept that one or more nucleotides is replaced by DNA, LNA, Morpholino,UNA, TNA, GNA, and/or FANA, etc.

As detailed below, substituting a single nucleotide at a given positionwith a modified version of the same nucleotide would produce a modifiedvariant (with 0 mismatches).

In another particular aspect, the RNAi agent comprises a sense strandcomprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3nt from the sense strand of any of the RNAi agents listed in the Tablesand modified and unmodified variants thereof.

RNAi agents to EPAS1 of the present disclosure can be used in RNAinterference.

Modifications of RNAi Agents

The present disclosure encompasses both unmodified and example modifiedRNAi agents, such as those disclosed in the Tables.

The present disclosure further encompasses any other modification of adisclosed RNAi agent (e.g., a modified variant).

For example, the disclosure encompasses a RNAi agent with a substitutionof a single nucleotide at a given position with a modified version ofthe same nucleotide. Thus a nucleotide (A, G, C or U) can be replaced bythe corresponding 5-fluorouracil, 5-bromouracil, 5-chlorouracil,5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3) w,or 2,6-diaminopurine.

Additional modified variants include the addition of any other moiety(e.g., a radiolabel or other tag or conjugate) to the RNAi agent;provided that the base sequence is identical, the addition of othermoieties produces a “modified variant” (with no mismatches).

Various sets of modifications can be used. These include the followingformats, which are used in various screens disclosed herein.

Two such sets of modifications include A51 S26 and A85S26; EPAS1 RNAiagents with these modification sets are presented in Table 5.

A51S53

Guide strand: All U and C except positions 1, 2 and 14 are 2′-OMe

Sense strand: All U as 2′-OMe-U.

A85S26 (“NEW”)

Guide strand: All U except position 1, 2 and 14 are 2′-OMe

Sense strand: All C and U are modified (2′-OMe)

However, other modifications of the EPAS1 RNAi agent sequences disclosedherein could be prepared. These include, as non-limiting examples:

A51 S26

A51: In guide strand all U as 2′-OMe-U and all C as 2′-OMe-C, exceptpos. 1, 2 and 14; 3′ overhangs as 2′-OMe-U 2′-OMe-U

S26: In sense strand all U as 2′-OMe-U and all C as 2′-OMe-C; 3′overhangs as 2′-OMe-U 2′-OMe-U

A22S26

A22 indicates that All UA as 2′-OMe-U A and all CA as 2′-OMe-C A

S26 indicates that All U as 2′-OMe-U and all C as 2′-OMe-C

All 3′ overhangs as 2′-OMe-U 2′-OMe-U

In addition to these modifications and patterns (e.g., formats) formodifications, other modifications or sets of modifications of thesequences provided can be generated using common knowledge of nucleicacid modification. These various aspects of the RNAi agents to EPAS1 ofthe present disclosure can be used in RNA interference.

RNA Interference

RNA interference (RNAi) is a post-transcriptional, targetedgene-silencing technique that uses double-stranded RNA (dsRNA) todegrade messenger RNA (mRNA) containing the same sequence as the dsRNA.The process of RNAi occurs when ribonuclease III (Dicer) cleaves thelonger dsRNA into shorter fragments called siRNAs. siRNAs interferingRNAs) produced by Dicer are typically about 21 to 23 nucleotides longand comprise about 19 base pair duplexes (though artificial siRNAs orRNAi agents can be shorter or longer, and/or blunt-ended, and/orcomprises one or more endcaps). The smaller RNA segments then mediatethe degradation of the target mRNA, Dicer has also been implicated inthe excision of 21- and 22-nucleotide small temporal RNAs (stRNAs) fromprecursor RNA of conserved structure that are implicated intranslational control. Hutvagner et al, 2001 Science 293: 834. The RNAiresponse also features an endonuclease complex, commonly referred to asan RNA-induced silencing complex (RISC), which mediates cleavage ofsingle-stranded mRNA complementary to the anti-sense strand of the RNAiagent. Cleavage of the target RNA takes place in the middle of theregion complementary to the anti-sense strand of the siRNA duplex.

In one aspect, an RNA interference agent includes a single-stranded RNAthat interacts with a target RNA sequence to direct the cleavage of thetarget RNA. Without wishing to be bound by theory, the presentdisclosure contemplates a long double-stranded RNA introduced intoplants and invertebrate cells is broken down into siRNA by a Type IIIendonuclease known as Dicer. Sharp et al. 2001 Genes Dev. 15:485. Dicer,a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pairshort interfering RNAs with characteristic two base 3′ overhangs.Bernstein, et al. 2001 Nature 409:363. The siRNAs are then incorporatedinto an RNA-induced silencing complex (RISC) where one or more helicasesunwind the siRNA duplex, enabling one of the now unpaired siRNA strandsto act as a “guide” strand to guide target recognition. Nykanen, et al.2001 Cell 107:309. Upon binding of the antisense guide strand to theappropriate target mRNA, one or more endonucleases within the RISCcleaves the target to induce silencing. Elbashir, et al. 2001 Genes Dev.15:188. Thus, in one aspect the present disclosure relates to asingle-stranded RNA that promotes the formation of a RISC complex toeffect silencing of the target gene.

Kits for RNAi synthesis are commercially available, e.g., from NewEngland Biolabs and Ambion.

A suitable RNAi agent can be selected by any process known in the art orconceivable by one of ordinary skill in the art. For example, theselection criteria can include one or more of the following steps:initial analysis of the EPAS1 gene sequence and design of RNAi agents;this design can take into consideration sequence similarity acrossspecies (human, cynomolgus, mouse, etc.) and dissimilarity to other(non-EPAS1) genes; screening of RNAi agents in vitro (e.g., at 10 nM inRKO cells); determination of EC50 in RKO cells; determination ofviability of cells treated with RNAi agents, including insensitive cellswhich do not require EPAS1 for survival, or sensitive cells, which dorequire EPAS1 for survival; testing with human PBMC (peripheral bloodmononuclear cells), e.g., to test levels of TNF-alpha to estimateimmunogenicity, wherein immunostimulatory sequences are less desired;testing in human whole blood assay, wherein fresh human blood is treatedwith an RNAi agent and cytokine/chemokine levels are determined [e.g.,TNF-alpha (tumor necrosis factor-alpha) and/or MCP1 (monocytechemotactic protein 1)], wherein Immunostimulatory sequences are lessdesired; determination of gene knockdown in vivo using subcutaneoustumors in test animals; EPAS1 target gene modulation analysis, e.g.using a pharmacodynamic (PD) marker, for example, other factors whoseexpression is affected by EPAS1, wherein EPAS1 knockdown leads to adose-dependent reduction of abundance of those components; andoptimization of specific modifications of the RNAi agents.

The dsRNA molecules (RNAi agents) described herein are thus useful inRNA interference of EPAS1.

Features of a RNAi Agent: Sense Strand, Antisense Strand and (Optional)Overhangs

In various aspects, the RNAi agents comprise a first strand and a secondstrand, e.g., a sense strand and an antisense strand (or an antisenseand a sense strand), optionally, either or both ends of either or bothstrand can comprise unpaired nucleotides (referred to herein as“overhangs”).

The term “antisense strand” refers to the strand of a RNAi agent whichincludes a region that is substantially complementary to a targetsequence. As used herein, the term “region of complementarity” refers tothe region on the antisense strand that is substantially complementaryto a sequence, for example a target sequence, as defined herein. Wherethe region of complementarity is not fully complementary to the targetsequence, the mismatches may be in the internal or terminal regions ofthe molecule. Generally, the most tolerated mismatches are in theterminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5 and/or3′ terminus.

The term “sense strand,” as used herein, refers to the strand of a RNAiagent that includes a region that is substantially complementary to aregion of the antisense strand as that term is defined herein.

The sequence of a gene may vary from individual to individual,especially at wobble positions within the coding segment, or in theuntranslated region; individuals may also differ from each other incoding sequence, resulting in additional differences in mRNA. Thesequence of the sense and antisense strands of the RNAi agent can thusbe designed to correspond to that of an individual patient, if and whereneeded. RNAi agents can also be modified in sequence to reduceimmunogenicity, binding to undesired mRNAs (e.g., “off-target effects”)or to increase stability in the blood. These sequence variants areindependent of chemical modification of the bases or 5′ or 3′ or otherend-caps of the RNAi agents.

The RNAi agents can also have overhangs of 0, 1, or 2 overhangs; in thecase of a 0 nt overhang, they are blunt-ended. A RNAi agent can thushave 0, 1 or 2 blunt ends. In a “blunt-ended RNAi agent” both strandsterminate in a base-pair; thus a blunt-ended molecule lacks either 3′ or5′ single-stranded nucleotide overhangs.

The RNAi agents can comprise overhang(s), blunt end(s), and/or 5′ and 3′endcap(s).

As used herein, the term “overhang” or “nucleotide overhang” refer to atleast one unpaired nucleotide that protrudes from the end of at leastone of the two strands of the duplex structure of a RNAi agent. Forexample, when a 3′-end of one strand of a dsRNA extends beyond the5′-end of the other strand, or vice versa, this forms a nucleotidicoverhang, e.g., the unpaired nucleotide(s) form the overhang. A dsRNAcan comprise an overhang of at least one nucleotide; alternatively theoverhang can comprise at least two nucleotides, at least threenucleotides, at least four nucleotides, at least five nucleotides ormore. An overhang can comprise or consist of a nucleotide/nucleosideanalog, including a deoxynucleotide/nucleoside. The overhang(s) may beon the sense strand, the antisense strand or any combination thereof.Furthermore, the nucleotide(s) of an overhang can be present on the 5′end, 3′ end or both ends of either an antisense or sense strand of adsRNA. The RNAi agent can also optionally comprise a cap. The term “Cap”and the like include a chemical moiety attached to the end of adouble-stranded nucleotide duplex, but is used herein to exclude achemical moiety that is a nucleotide or nucleoside. A “3 Cap” isattached at the 3′ end of a nucleotide or oligonucleotide and protectsthe molecule from degradation, e.g., from nucleases, such as those inblood serum or intestinal fluid. A non-nucleotidic 3′ cap is not anucleotide and can replace a TT or UU dinucleotide at the end of ablunt-ended RNAi agent. In one aspect, non-nucleotidic 3′ end caps areas disclosed in, for example, WO 2005/021749 and WO 2007/128477; andU.S. Pat. Nos. 8,097,716; 8,084,600; and 8,344,128. A “5′ cap” isattached at the 5′ end of a nucleotide or oligonucleotide. A cap shouldnot interfere (or unduly interfere) with RNAi activity.

The present disclosure thus contemplates a RNAi agent specific to EPAS1comprising an antisense strand (which may be contiguous or connected viaa linker or loop) in a RNAi agent. In a more specific aspect, an RNAiagent comprises an antisense strand and a sense strand which togethercomprise a double-stranded or complementary region. In one aspect, itcan also optionally comprise one or two overhangs and/or one or twocaps. The RNAi agent is used to induce RNA interference of the targetgene, EPAS1.

Target and Complementary Sequences

The RNAi agents of the present disclosure target (e.g., specificallybind to, anneal to, etc.) the mRNA encoding EPAS1. The use of the RNAiagent specific to EPAS1 results in a decrease of EPAS1 activity, leveland/or expression. Particularly in one aspect, in the case of a diseasestate characterized by over-expression or hyper-activity of EPAS1,administration of a RNAi agent to EPAS1 knocks down the EPAS1 geneenough to restore a normal level of EPAS1 activity or expression.

In one aspect, the first or second strand of the RNAi comprises asequence complementary to that of the target nucleic acid, EPAS1.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

As used herein, “target sequence” or “target gene” refer to a contiguousportion of the nucleotide sequence of an mRNA molecule formed during thetranscription, of a gene, EPAS1 gene, including mRNA that is a productof RNA processing of a primary transcription product. The target portionof the sequence will be at least long enough to serve as a substrate forRNAi-directed cleavage at or near that portion. For example, the targetsequence will generally be from 9-36 nucleotides (“nt”) in length, e.g.,15-30 nt in length, including all sub-ranges therebetween. Asnon-limiting examples, the target sequence can be from 15-30 nt, 15-26nt, 15-23 nt, 15-22 nt, 15-21 nt, 15-20 nt, 15-19 nt, 15-18 nt, 15-17nt, 18-30 nt, 18-26 nt, 18-23 nt, 18-22 nt, 18-21 nt, 18-20 nt, 19-30nt, 19-26 nt, 19-23 nt, 19-22 nt, 19-21 nt, 19-20 nt, 19 nt, 20-30 nt,20-26 nt, 20-25 nt, 20-24 nt, 20-23 nt, 20-22 nt, 20-21 nt, 20 nt, 21-30nt, 21-26 nt, 21-25 nt, 21-24 nt, 21-23 nt, or 21-22 nt, 21 nt, 22 nt,or 23 nt. The sense and antisense strands of the RNAi comprise asequence complementary to that of the target nucleic acid, EPAS1.

As used herein, and unless otherwise indicated, the term “complementary”refers to the ability of an oligonucleotide or polynucleotide comprisinga first nucleotide sequence to hybridize and form a duplex structureunder certain conditions with an oligonucleotide or polynucleotidecomprising a second nucleotide sequence. Such conditions can, forexample, be stringent, e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA,50° C. or 70° C. for 12-16 hours followed by washing. Other conditions,such as physiologically relevant conditions as may be encountered insidean organism, can apply. The skilled person will be able to determine theset of conditions most appropriate for a test of complementarity of twosequences in accordance with the ultimate application of the hybridizednucleotides.

Complementary sequences within a RNAi agent, e.g., within a dsRNA asdescribed herein, include base-paired oligonucleotides orpolynucleotides comprising a first nucleotide sequence to anoligonucleotide or polynucleotide comprising a second nucleotidesequence over the entire length of one or both nucleotide sequences.Such sequences can be referred to as “fully complementary” with respectto each other herein. However, where a first sequence is referred to as“substantially complementary” with respect to a second sequence herein,the two sequences can be fully complementary, or they may form one ormore, but generally not more than 5, 4, 3 or 2 mismatched base pairsupon hybridization for a duplex up to 30 base pairs, while retaining theability to hybridize under the conditions most relevant to theirultimate application, e.g., inhibition of gene expression via a RISCpathway. However, where two oligonucleotides are designed to form, uponhybridization, one or more single-stranded overhangs, such overhangsshall not be regarded as mismatches with regard to the determination ofcomplementarity. For example, a dsRNA comprising one oligonucleotide 21nucleotides in length and another oligonucleotide 23 nucleotides inlength, wherein the longer oligonucleotide comprises a sequence of 21nucleotides that is fully complementary to the shorter oligonucleotide,may yet be referred to as “fully complementary” for the purposesdescribed herein. The term “overhang” describes an unpaired nucleotideat the 3′ or 5′ end of a double-stranded nucleotide duplex, as describedabove. In one aspect, the overhang is 1 to 4 nt long and is on the 3′end.

“Complementary” sequences, as used herein, may also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in as far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs includes, but are not limited to,Wobble or Hoogstein base pairing. The terms “complementary,” “fullycomplementary” and “substantially complementary” herein may furthermorebe used with respect to the base matching between the sense strand andthe antisense strand of a dsRNA, or between the antisense strand of aRNAi agent and a target sequence, as will be understood from the contextof their use. As used herein, a polynucleotide that is “substantiallycomplementary to at least part of” a messenger RNA (mRNA) refers to apolynucleotide that is substantially complementary to a contiguousportion of the mRNA of interest (e.g., an mRNA encoding EPAS1). Forexample, a polynucleotide is complementary to at least a part of a EPAS1mRNA if the sequence is substantially complementary to a non-interruptedportion of an mRNA encoding EPAS1.

Thus, the RNAi agent of the present disclosure is complimentary orsubstantially complimentary to a target sequence in the target EPAS1 andis double-stranded, comprising a sense and an antisense strand (whichcan be contiguous, linked via a loop, or otherwise joined), where thedouble-stranded region an be 9 to 36 bp long (particularly for example,19-22 bp or 19-23 bp long), and can furthermore optionally comprise a 3′or 5′ overhang, and the RNAi agent can furthermore comprise a 3′ cap.The RNAi agent mediates RNA interference, down-regulating or inhibitingthe level, expression and/or activity of EPAS1, and/or establishing orre-establishing an approximately normal level of EPAS1 and/or EPAS1activity, or other biological function related to EPAS1.

Thus, the RNAi agent of the present disclosure is complimentary orsubstantially complimentary to a target sequence in the target EPAS1 andis double-stranded, comprising a sense and an antisense strand (whichcan be contiguous, linked via a loop, or otherwise joined), where thedouble-stranded region an be 9 to 36 bp long (particularly for example,19-22 bp or 19-23 bp long), and can furthermore optionally comprise a 3′or 5′ overhang, and the RNAi agent can furthermore comprise a 3′ cap.The RNAi agent mediates RNA interference, down-regulating or inhibitingthe level, expression and/or activity of EPAS1, and/or establishing orre-establishing an approximately normal level of EPAS1 activity orexpression.

The term “double-stranded RNA” or “dsRNA,” as used herein, refers to anRNAi agent comprising a first and a second strand; e.g., a compositionthat includes an RNA molecule or complex of molecules having ahybridized duplex region that comprises two anti-parallel andsubstantially complementary nucleic acid strands, which will be referredto as having “sense” and “antisense” orientations with respect to atarget RNA. The antisense strand, with respect to the mRNA target, isalso called the “guide” strand, and the sense strand is also called the“passenger” strand. As used herein, depending on the context, the“first” strand can be the guide of antisense strand, and the “second”strand can be the passenger or sense strand. Also as used herein, againdepending on the context, the “first” strand can be the passenger orsense strand, and the “second” strand can be the guide or antisense. Thepassenger strand can include at least one or more of the following: oneor more extra nucleotides (e.g., a bulge or 1 nt loop) compared to theother strand, a nick, a gap, etc., compared to the other strand. Invarious aspects, the first strand is the sense strand and the secondstrand is the anti sense strand. In other aspects, the first strand isthe anti-sense strand, and the second strand is the sense strand.

The duplex region can be of any length that permits loading into theRISC complex and subsequent specific degradation of a desired target RNAthrough a RISC pathway, but will typically range from 9 to 36 base pairs(“bp”) in length, e.g., 15-30 base pairs in length. Considering a duplexbetween 9 and 36 base pairs, the duplex can be any length in this range,for example, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 bp, and anysub-range therebetween, including, but not limited to 15-30 base pairs,15-26 bp, 15-23 bp, 15-22 bp, 15-21 bp, 15-20 bp, 15-19 bp, 15-18 bp,15-17 bp, 18-30 bp, 18-26 bp, 18-23 bp, 18-22 bp, 18-21 bp, 18-20 bp,19-30 bp, 19-26 bp, 19-23 bp, 19-22 bp, 19-21 bp, 19-20 bp, 19 bp, 20-30bp, 20-26 bp, 20-25 bp, 20-24 bp, 20-23 bp, 20-22 bp, 20-21 bp, 20 bp,21-30 bp, 21-26 bp, 21-25 bp, 21-24 bp, 21-23 bp, 21-22 bp, 21 bp, 22bp, or 23 bp.

The dsRNAs generated in the cell by processing with Dicer and similarenzymes are generally in the range of about 19 to about 22 base pairs inlength, although artificial RNAi agents can be synthesized or made byany method known in the art. One strand of the duplex region of a dsRNAcomprises a sequence that is substantially complementary to a region ofa target RNA. The two strands forming the duplex structure can be from asingle RNA molecule having at least one self-complementary duplexregion, or can be formed from two or more separate RNA molecules thathybridize to form the duplex. Where the duplex region is formed from twoself-complementary regions of a single molecule, molecule can have aduplex region separated by a single stranded chain of nucleotides(herein referred to as a “hairpin loop”, e.g., such as found in an shRNAconstruct) between the 3′-end of one strand and the 5′-end of therespective other strand forming the duplex structure. The hairpin loopcan comprise at least one unpaired nucleotide; in some aspects thehairpin, loop can comprise at least 3, at least 4, at least 5, at least6, at least 7, at least 8, at least 9, at least 10, at least 20, atleast 23 or more unpaired nucleotides. Where the two substantiallycomplementary strands of a dsRNA are comprised by separate RNAmolecules, those molecules need not, but can be covalently connected.Where the two strands are connected covalently by a hairpin loop, theconstruct is generally referred to herein and in the art as a “shRNA”.Where the two strands are connected covalently by means other than ahairpin loop, the connecting structure is referred to as a “linker.” Theterm “siRNA” is also used herein to refer to a dsRNA as described above.

RNAi Agents Lowering or Normalizing EPAS1 Level, Expression and/orActivity

RNAi agents for targeting EPAS1 include those which bind to a EPAS1sequence provided herein and which work to reduce EPAS1 through a RNAimechanism. Example RNAi agents (e.g., siRNAs) to EPAS1 are provided,e.g., Table 1.

Any method known in the art can be use to measure changes in EPAS1activity, level, and/or expression induced by a EPAS1 RNAi agent.Measurements can be performed at multiple timepoints, prior to, duringand after administration of the RNAi agent, to determine the effect ofthe RNAi agent.

The RNAi agents of the present disclosure silence, inhibit theexpression of, down-regulate the expression of, and/or suppress theexpress on of EPAS1, such that an approximately normal level of EPAS1activity or expression is restored.

In addition, in various aspects, depending on the disease condition andbiological context, it is acceptable to use the RNAi agents of thepresent disclosure to establish a level of EPAS1 expression, activityand/or level which is below the normal level, or above the normal level,depending on the therapeutic outcome that is desired.

Any method known in the art can be use to measure changes in EPAS1activity, level and/or expression induced by a EPAS1 siRNA. Measurementscan be performed at multiple timepoints, prior to, during and afteradministration of the siRNA determine the effect of the siRNA.

The terms “silence,” “inhibit the expression of,” “down-regulate theexpression of,” “suppress the expression of,” and the like, in so far asthey refer to a EPAS1 gene, herein refer to the at least partialsuppression of the expression of a EPAS1 gene, as manifested by areduction of the amount of EPAS1 mRNA which may be isolated from ordetected in a first cell or group of cells in which a EPAS1 gene istranscribed and which has or have been treated such that the expressionof a EPAS1 gene is inhibited, as compared to a second or group of cellssubstantially identical to first cell or group of cells but which has ornot been so treated (control cells). The degree of inhibition is usuallyexpressed in terms of

$\begin{matrix}{{\frac{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right) - \left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{treated}\mspace{14mu}{cells}} \right)}{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right)} \cdot 100}\%} & {{Equation}\mspace{14mu} 1}\end{matrix}$

Alternatively, the degree of inhibition may be given in terms of areduction of a parameter that is functionally linked to EPAS1 geneexpression, e.g.; the amount of protein encoded by a EPAS1 gene,alteration in expression of a protein whose expression is dependent onEPAS1, etc. In principle, EPAS1 gene silencing may be determined in anycell expressing EPAS1, either constitutively or by genomic engineering,and by any appropriate assay. However, when a reference or control isneeded in order to determine whether a given RNAi agent inhibits theexpression of EPAS1 by a certain degree and therefore is encompassed bythe instant disclosure, the assays provided in the Examples below shallserve as such reference.

For example, in certain instances, expression of EPAS1 is suppressed byat least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% byadministration of a RNAi agent featured in the present disclosure. Insome aspects. EPAS1 is suppressed by at least about 60%, 70%, or 80% byadministration of a RNAi agent featured in the present disclosure. Insome aspects, EPAS1 is suppressed by at least about 85%, 90%, or 95% ormore by administration of a RNAi agent, as described herein. In oneaspect, the degree of EPAS1 suppression is determined by loss of fulllength EPAS1 mRNA in a treated cell compared to an untreated cell. Inone aspect, the degree of EPAS1 suppression is determined with aphenotypic assay that monitors loss of proliferative activity and/orcell death. Other aspects are as provided in the Examples.

A suitable RNAi agent can be selected by any process known in the art orconceivable by one of ordinary skill in the art. For example, theselection criteria can include one or more of the following steps:initial analysis of the EPAS1 gene sequence and design of RNAi agents;this design can take into consideration sequence similarity acrossspecies (human, cynomolgus, mouse, etc.) and dissimilarity to other(non-EPAS1) genes; screening of RNAi agents in vitro (e.g., at 10 nM and1 nM in 786-O cells): selection of RNAi agents with high knock-down at10 nM and 1 nM in 786-O cells; determination of EC50 in 786-O cells;confirmation of EC50 in a RCC cell line (786-O cells): analysis of alack of effect on cell growth relative to a control siRNA; reduction in786-O cells of expression of a HRE-luc (luciferase) reporter gene andnot control UB6-luc (luciferase) reporter gene; Western blots to measureHif-1, Hif-2, and ARNT levels; testing with human PBMC (peripheral bloodmononuclear cells), e.g., to test levels of TNF-alpha to estimateimmunogenicity, wherein immunostimulatory sequences are less desired;testing in human whole blood assay, wherein fresh human blood is treatedwith an RNAi agent and cytokine/chemokine levels are determined [e.g.,TNF-alpha (tumor necrosis factor-alpha) and/or MCP1 (monocytechemotactic protein 1)], wherein immunostimulatory sequences are lessdesired; determination of gene knockdown in vivo using subcutaneoustumors in test animals; EPAS1 target gene modulation analysis, e.g.,using a pharmacodynamic (PD) marker, for example, EGLN3, SLC2A1 andVEGF, wherein EPAS1 knockdown leads to a dose-dependent alteration ofEGLN3, SLC2A1 and VEGF expression in cells; and optimization of specificmodifications of the RNAi agents. As appropriate, other cell lines canbe used in place of those listed above to identify RNAi agents capableof lowering EPAS1 levels or decrease symptoms of a EPAS1-relateddisease.

By “lower” in the context of EPAS1 or a symptom of a EPAS1-relateddisease is meant a statistically significant decrease in such level. Thedecrease can be, for example, at least 10%, at least 20%, at least 30%,at least 40% or more. If, for a particular disease, or for an individualsuffering from a particular disease, the levels or expression of EPAS1are elevated, treatment with a EPAS1 RNAi agent of the presentdisclosure can particularly reduce the level or expression of EPAS1 to alevel considered in the literature as within the range of normal for anindividual without such disorder, or to a level that reduces orameliorates symptoms of a disease. The level or expression of EPAS1 canbe measured by evaluation of mRNA (e.g., via Northern blots or PCR), orprotein (e.g., Western blots). The effect of a RNAi agent on EPAS1expression can be determined by measuring EPAS1 gene transcription rates(e.g., via Northern blots; or reverse transcriptase polymerase chainreaction or real-time polymerase chain reaction).

As used herein, “down-regulates” refers to any statistically significantdecrease in a biological activity and/or expression of EPAS1, includingfull blocking of the activity (i.e., complete inhibition) and/orexpression. For example, “down-regulation” can refer to a decrease of atleast about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% in EPAS1 level,activity and/or expression.

As used herein, the term “inhibit” or “inhibiting” EPAS1 refers to anystatistically significant decrease in biological level, activity and/orexpression of EPAS1, including full blocking of the activity and/orexpression. For example, “inhibition” can refer to a decrease of atleast about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% in EPAS1 level,activity and/or expression. As used herein, the term “inhibit” similarlyrefers to a significant decrease in level, activity and/or expression,while referring to any other biological agent or composition.

By “level”, it is meant that the EPAS1 RNAi agent can alter the level ofEPAS1, e.g., the level of EPAS1 mRNA or the level of EPAS1 protein, orthe level of activity of EPAS1.

Some diseases include any EPAS1-related disease disclosed herein orknown in the literature. Particularly in one aspect, in the case of adisease characterized by over-expression and or hyper-activity of EPAS1,administration of a RNAi agent to EPAS1 reduces the level, expressionand/or activity of EPAS1. Thus, in various aspects, administration of aRNAi agent to EPAS1 particularly establishes or re-establishes a normalor approximately normal level of EPAS1 activity, expression and/orlevel.

By “normal” or “approximately normal” in terms of level, expressionand/or activity, is meant at least; about 50%, about 60%, about 70%,about 80%, about 90%, and/or about 100%; and/or no more than: about100%, about 120%, about 130%, about 140%, or about 150% of the level,expression or activity of EPAS1 in a healthy cell, tissue, or organ.This can be measured using, for example, lung or kidney homogenates, asdescribed in Gambling et al. 2 Kidney Intl. 65: 1774-1781. Particularlyin one aspect, administration of the appropriate amount of theappropriate EPAS1 RNAi agent restores EPAS1 level, activity and/orexpression to about 50% to about 150%, more particularly about 60% toabout 140%, more particularly to about 70% to about 130%, moreparticularly to about 80% to about 120%, more particularly to about 90%to about 110%, and most particularly to about 100% of that of a healthycell, tissue or organ. Administration of a EPAS1 RNAi to a patient witha EPAS1-related disease thus particularly restores the level, activity,and/or expression of EPAS1 and the level of Na⁺ reabsorption to anapproximately normal level, as determined by direct measurements ofEPAS1 mRNA or protein levels, or indirect determinations. In addition,the preferred target amount of EPAS1 level, expression and/or activityafter EPAS1 RNAi agent administration can be calculated to take intoaccount any other perturbations in a EPAS1-related pathway. For example,if another factor in a EPAS1-related pathway is either over- orunder-expressed, EPAS1 level, expression or activity may be modulated toattain a more normal state.

In addition, various aspects, depending on the disease condition andbiological context, it is acceptable to use the RNAi agents of thepresent disclosure to establish a level of EPAS1 expression, activityand/or level which is below the normal level, or above the normal level.

Types of RNAi Agents and Modification Thereof

The use of RNAi agents or compositions comprising an antisense nucleicacid to down-modulate the expression of a particular protein in a cellis well known in the art. A RNAi agent comprises a sequencecomplementary to, and is capable of hydrogen bonding to, the codingstrand of another nucleic acid (e.g., an mRNA). Thus, in variousaspects, the RNAi agents of the present disclosure encompass any RNAiagents which target (e.g., are complementary, capable of hybridizing orhydrogen bonding to, etc.) any sequence presented, e.g., in any of theTables.

Once a functional guide strand has been identified, many variations tothe guide and/or passenger strand can be made. For example, the RNAiagent may have modifications internally, or at one or both ends. Themodifications at the ends can help stabilize the RNAi agent, protectingit from degradation by nucleases in the blood. The RNAi agents mayoptionally be directed to regions of the EPAS1 mRNA known or predictedto be near or at splice sites of the gene.

A RNAi agent can be constructed using chemical synthesis and enzymaticligation reactions using procedures known in the art. For example, RNAiagent can be chemically synthesized using naturally-occurringnucleotides or variously modified nucleotides designed to decreaseoff-target effects, and/or increase the biological stability of themolecules or to increase the physical stability of the duplex formedbetween the antisense and sense nucleic acids, e.g., phosphorothioatederivatives and acridine substituted nucleotides can be used.

“G,” “C” “A,” “T” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, thymidine and uracil as a base,respectively. However, the terms “ribonucleotide”, “deoxynucleotide”, or“nucleotide” can also refer to a modified nucleotide or a surrogatereplacement moiety. The skilled person is well aware that guanine,cytosine, adenine, and uracil may be replaced by other moieties withoutsubstantially altering the base pairing properties of an oligonucleotidecomprising a nucleotide bearing such replacement moiety. For example,without limitation, a nucleotide comprising inosine as its base may basepair with nucleotides containing adenine, cytosine, or uracil. Hence,nucleotides containing uracil, guanine, or adenine may be replaced inthe nucleotide sequences of dsRNA featured in the present disclosure bya nucleotide containing, for example, inosine. In another example,adenine and cytosine anywhere in the oligonucleotide can be replacedwith guanine and uracil, respectively to form Wobble base pairing withthe target mRNA. Sequences containing such replacement moieties aresuitable for the compositions and methods featured in the presentdisclosure.

The skilled artisan will recognize that the term “RNA molecule” or“ribonucleic acid molecule” encompasses not only RNA molecules asexpressed or found in nature (i.e., are naturally occurring), but alsonon-naturally occurring analogs and derivatives of RNA comprising one ormore ribonucleotide/ribonucleoside analogs or derivatives as describedherein or as known in the art. The RNA can be modified in the nucleobasestructure or in the ribose-phosphate backbone structure, e.g., asdescribed herein below. However, the molecules comprising ribonucleosideanalogs or derivatives must retain the ability to form a duplex. Asnon-limiting examples, either or both strand of an RNAi agent cancomprise at least one modified ribonucleoside, including but not limitedto a 2′-O-methyl modified nucleotide, a nucleoside comprising a 5′phosphorothioate group, a terminal nucleoside linked to a cholesterylderivative or dodecanoic acid bisdecylamide group, a locked nucleoside,an abasic nucleoside, a 2′-deoxy-2′-fluoro modified nucleoside, a2′-amino-modified nucleoside, 2′-alkyl-modified nucleoside, morpholinonucleoside, an unlocked ribonucleotide (e.g., an acyclic nucleotidemonomer, as described in WO 2008/147824), a phosphoramidate or anon-natural base comprising nucleoside, or any combination thereof.Alternatively, an RNA molecule can comprise at least two modifiedribonucleosides, at least 3, at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9, at least 10, at least 15, at least 20or more, up to the entire length of the dsRNA molecule. Themodifications need not be the same for each of such a plurality ofmodified ribonucleosides in an RNA molecule. In one aspect, modifiedRNAs contemplated for use in methods and compositions described hereinare peptide nucleic acids (PNAs) that have the ability to form therequired duplex structure and that permit or mediate the specificdegradation of a target RNA via a RISC pathway. In addition, the RNAiagent can comprise one or two strands which are a RNA, or a mixture ofRNA, DNA, LNA, Morpholino, UNA, TNA, GNA, and/or FANA, modified RNA,etc. As a non-limiting example, one or both strands could be, forexample, RNA except that one or more nucleotides is replaced by DNA,LNA, Morpholino, UNA, TNA, GNA, and/or FANA, and/or modified RNA (e.g.,any modified RNA disclosed herein or known in the art, such as 2′modified RNA, including but not limited to 2′-F, 2′-OMe. 2′-MOE RNA,etc.).

Examples of modified nucleotides which can be used to generate the RNAiagent include 5-fluorouracil, 5-bromouracil, 5-chlorouracil,5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil.5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. These and other example modifications are shownin FIG. 1, herein, and Usman et al. 1992 TIBS 17:34; Usman et al. 1994Nucl. Acids Symp. Ser. 31: 163; Burgin et al. 1996 Biochem. 35: 14090.

A “modified variant” of a sequence disclosed herein includes any variantcomprising the same sequence, but with a modification in the base,sugar, phosphate or backbone (but not a base substitution, e.g., A forG, or C for U). Thus, a modified variant can comprise any modifiednucleotide described above (e.g., 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil, etc.). When a base is replaced by acorresponding modified base (e.g., A for modified A), these modifiednucleotides do not constitute a mismatch or base difference. Thus agiven sequence with a U at a particular position and a modified variantcomprising a 5-fluorouracil, 5-bromouracil, 5-chlorouracil, or5-iodouracil at the same sequence would differ by 0 nt (or have nomismatches); however, a given sequence with a C at a particular positionand a different sequence with a 5-fluorouracil (wherein the twosequences are otherwise identical) would differ by 1 nt (1 mismatch).

Replacing the 3′-terminal nucleotide overhanging segments of a 21-mersiRNA duplex having two-nucleotide 3′-overhangs withdeoxyribonucleotides does not have an adverse effect on RNAi activity.Replacing up to four nucleotides on each end of the siRNA withdeoxyribonucleotides has been well tolerated, whereas completesubstitution with deoxyribonucleotides results in no RNAi activity.International PCT Publication No. WO 00/44914, and Beach et al.International PCT Publication No. WO 01/68836 preliminarily suggest thatsiRNA may include modifications to either the phosphate-sugar backboneor the nucleoside to include at least one of a nitrogen or sulfurheteroatom, Kreutzer et al. Canadian Patent Application No. 2,359,180,also describe certain chemical modifications for use in dsRNA constructsin order to counteract activation of double-stranded RNA-dependentprotein kinase PKR, specifically 2′-amino or 2′-O-methyl nucleotides,and nucleotides containing a 2′-O or 4′-C methylene bridge. Additional3′-terminal nucleotide overhangs include dT (deoxythimidine), 2′-C),4′-C-ethylene thymidine (eT), and 2-hydroxyethyl phosphate (hp).4-thiouracil and 5-bromouracil substitutions can also be made. Parrishet al. 2000 Molecular Cell 6: 1077-1087.

Those skilled in the art will appreciate that it is possible tosynthesize and modify the siRNA as desired, using any conventionalmethod known in the art (see Henschel et al. 2004 DEQOR: a web-basedtool for the design and quality control of siRNAs. Nucleic AcidsResearch 32 (Web Server Issue): W113-W120). In addition, if the RNAiagent is a shRNA, it will be apparent to those skilled in the art thatthere are a variety of regulatory sequences (for example, constitutiveor inducible promoters, tissue-specific promoters or functionalfragments thereof, etc.) which are useful for shRNA expressionconstruct/vector.

There are several examples in the art describing sugar, base, phosphateand backbone modifications that can be introduced into nucleic acidmolecules with significant enhancement in their nuclease stability andefficacy. For example, oligonucleotides are modified to enhancestability and/or enhance biological activity by modification withnuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-flouro,2′-O-methyl, 2′-O-allyl, 2′-H, nucleotide base modifications (for areview sec Usman and Cedergren 1992 TIBS. 17: 34; Usman et al. 1994Nucleic Acids Symp. Ser. 31; 163; Burgin et al. 1996 Biochemistry 35:14090). Sugar modification of nucleic acid molecules are extensivelydescribed in the art.

In various aspects, the RNAi agent comprises a 2′-modification selectedfrom the group consisting of: 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-methyl,2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), and2′-O—N-methylacetamido (2′-O-NMA).

Additional modifications and conjugations of RNAi agents have beendescribed. Soutschek et al. 2004 Nature 432: 173-178 presentedconjugation of cholesterol to the 3′-end of the sense strand of a siRNAmolecule by means of a pyrrolidine linker, thereby generating a covalentand irreversible conjugate. Chemical modifications (includingconjugation with other molecules) of RNAi agents may also be made toimprove the in vivo pharmacokinetic retention time and efficiency.

In various aspects, the RNAi agent to EPAS1 comprises at least one5′-uridine-adenine-3′ (5′-ua-3′) dinucleotide, wherein the uridine is a2′-modified nucleotide; at least one 5′-uridine-guanine-3′ (5′-ug-3′)dinucleotide, wherein the 5′-uridine is a 2′-modified nucleotide; atleast one 5′-cytidine-adenine-3′ (5′-ca-3′) dinucleotide, wherein the5′-cytidine is a 2′-modified nucleotide; and/or at least one5′-uridine-uridine-3′ (5′-uu-3′) dinucleotide, wherein the 5′-uridine isa 2′-modified nucleotide. In certain aspects, the RNAi agent cancomprise a non-natural nucleobase, wherein the non-natural nucleobase isdifluorotolyl, nitroindolyl, nitropyrrolyl, or nitroimidazolyl. In aparticular aspect, the non-natural nucleobase is difluorotolyl. Incertain aspects, only one of the two oligonucleotide strands contains anon-natural nucleobase. In certain aspects, both of the oligonucleotidestrands contain a non-natural nucleobase.

In another aspect, the RNAi comprises a gap or contains mismatchcomprising an abasic nucleotide.

In another aspect, the RNAi agent has a single-stranded nick (e.g., abreak or missing bond in the backbone). In various aspects, asingle-stranded nick can be in either the sense or anti-sense strand, orboth.

This nick can be, for example, in the sense strand, producing a smallinternally segmented interfering RNA, or sisiRNA, which may have lessoff-target effects than the corresponding RNAi agent without a nick.See, for example, WO 2007/107162 to Wengels and Kjems.

The antisense nucleic acid or RNAi agent can also have an alternativebackbone such as locked nucleic acids (LNA). Morpholinos, peptidicnucleic acids (PNA), threose nucleic acid (TNA), or glycol nucleic acid(GNA), or FANA and/or it can be labeled (e.g., radiolabeled or otherwisetagged). FANA are described in Dowler et al. 2006 Nucl. Acids Res. 34:1669-1675.

One or both strands can comprise an alternative backbone.

In yet another aspect, the RNAi agent employed by the methods of thepresent disclosure can include an α-anomeric nucleic acid molecule. Anα-anomeric nucleic acid molecule forms specific double-stranded hybridswith complementary RNA in which, contrary to the usual β-units, thestrands run parallel to each other. Gaultier et al. 1987 Nucleic Acids.Res. 15:6625-6641.

The antisense nucleic acid molecule can also comprise a5′-o-methylribonucleotide (Inoue et al. 1987 Nucleic Acids Res. 15;6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. 1987 FEBS Lett.215: 327-330).

Other modifications and/or other changes can be made to the RNAi agent.A portion of the RNAi agent can be double-stranded DNA, while anotherportion is double-stranded RNA, forming a DNA-RNA chimera (See, forexample, Yamato et al. 2011. Cancer Gene Ther. 18: 587-597). Mismatchesbetween the guide and passenger stand can also be introduced, thoughsome positions may be better suited than others (Sec, for example, U.S.Patent App. No. 2009/0209626 to Khvorova). The passenger strand can alsobe shortened, to as short as 15 or 16 nt, while the guide strand remains19 nt or longer (See, for example. Sun et al. 2008 Nature Biotech. 26:1379-1382; and Chu and Rana 2008 RNA 14: 1714-1719). This can increaseincorporation of the guide strand into the RNA-induced Silence Complex(RISC), and decrease incorporation of the passenger strand, thanreducing off-target effects. In some cases, the passenger strand may bemore amenable to modification (e.g., single-stranded nicking, nucleotidemodifications, and shortening) than the guide strand.

These and many other modifications can be made once a functional guidestrand is identified.

Pharmaceutical Compositions of RNAi Agents

As used here, a “pharmaceutical composition” comprises apharmaceutically effective amount of one or more EPAS1 RNAi agent, apharmaceutically acceptable carrier, and, optionally, an additionaldisease treatment which works synergistically with the RNAi agent. Asused herein, “pharmacologically effective amount,” “therapeuticallyeffective amount” or simply “effective amount” refers to that amount ofa RNAi agent effective to produce the intended pharmacological,therapeutic or preventive result. For example, if a given clinicaltreatment is considered effective where there is at least a 10%reduction in a measurable parameter associated with a disease ordisorder, a therapeutically effective amount of a drug for the treatmentof that disease or disorder is the amount necessary to effect at least a10% reduction in that parameter. In this aspect, a therapeuticallyeffective amount of a RNAi agent targeting EPAS1 can reduce EPAS1protein levels by at least 10%. In additional aspects, a given clinicaltreatment is considered effective where there is at least a 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% reduction in ameasurable parameter associated with a disease or disorder, and thetherapeutically effective amount of a drug for the treatment of thatdisease or disorder is the amount necessary to effect at least a 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% reduction,respectively, in that parameter:

The term “pharmaceutically acceptable carrier” refers to a carrier foradministration of a therapeutic agent. Such carriers include, but arenot limited to, lipid nanoparticles, saline, buffered saline, dextrose,water, glycerol, ethanol, and combinations thereof. The termspecifically excludes cell culture medium. Any appropriatepharmaceutical carrier known in the art can be used in conjunction withthe RNAi agents disclosed herein.

Pharmaceutical Composition Comprising a RNAi Agent to EPAS1

Additional components of a pharmaceutical composition comprising a RNAiAgent to EPAS1 are contemplated to aid in delivery, stability, efficacy,or reduction of immunogenicity.

Liposomes have been used previously for drug delivery (e.g., delivery ofa chemotherapeutic). Liposomes (e.g., cationic liposomes) are describedin PCT publications WO02/100435A1, WO03/015757A1, WO04029213A2; andWO/2011/076807; U.S. Pat. Nos. 5,962,016; 5,030,453; and 6,680,068, andU.S. Patent Application 2004/0208921. A process of making liposomes isalso described in WO04/002453A1. Furthermore, neutral lipids have beenincorporated into cationic liposomes (e.g., Farhood et al, 1995), aswell as PEGylated lipids.

Cationic liposomes have been used to deliver RNAi agent to various celltypes (Sioud and Sorensen 2003; U.S. Patent Application 2004/0204377,Duxbury et al., 2004; Donze and Picard, 2002).

Use of neutral liposomes disclosed in Miller et al. 1998, and U.S.Patent Application 2003/0012812.

As used herein, the term “SNALP” refers to a stable nucleic acid-lipidparticle. A SNALP represents a vesicle of lipids coating a reducedaqueous interior comprising a nucleic acid such as an iRNA or a plasmidfrom which an iRNA is transcribed. SNALPs are described, e.g., in U.S.Patent Application Publication Nos. 20060240093, 20070135372, and inInternational Application No. WO 2009082817.

Chemical transfection using lipid-based, amine-based and polymer-basedtechniques is disclosed in products from Ambion Inc., Austin, Tex.; andNovagen, EMD Biosciences, Inc. an Affiliate of Merck KGaA, Darmstadt,Germany); Ovcharenko D (2003) “Efficient delivery of siRNAs to humanprimary cells.” Ambion TechNotes 10 (5): 15-16). Additionally, Song etal. (Nat Med. published online (Feb. 10, 2003) doi: 10.1038/nm828) andothers [Caplen et al. 2001 Proc. Natl. Acad. Sci. (USA), 98: 9742-9747;and McCaffrey et al. Nature 414: 34-39] disclose that liver cells can beefficiently transfected by injection of the siRNA into a mammal'scirculatory system.

A variety of molecules have been used for cell-specific RNAi agentdeliver). See, for example, WO/2011/076807. For example, the nucleicacid-condensing property of protamine has been combined with specificantibodies to deliver siRNAs. Song et al. 2005 Nat Biotech. 23: 709-717.The self-assembly PEGylated polycation polyethylenimine (PEI) has alsobeen used to condense and protect siRNAs. Schiffelers et al. 2004 Nucl.Acids Res. 32: e149, 141-110.

The RNAi agents of the present disclosure can be delivered via, forexample, Lipid nanoparticles (LNP); neutral liposomes (NL); polymernanoparticles; double-stranded RNA binding motifs (dsRBMs); or viamodification of the RNAi agent (e.g., covalent attachment to the dsRNA)or by any method known in the art for delivery of a RNAi agentcomprising nucleic acids.

Lipid nanoparticles (LNP) are self-assembling cationic lipid basedsystems. These can comprise, for example, a neutral lipid (the liposomebase); a cationic lipid (for siRNA loading); cholesterol (forstabilizing the liposomes); and PEG-lipid (for stabilizing theformulation, charge shielding and extended circulation in thebloodstream).

The cationic lipid can comprise, for example, a headgroup, a linker, atail and a cholesterol tail. The LNP can have, for example, good tumordelivery, extended circulation in the blood, small particles (e.g., lessthan 100 nm), and stability in the tumor microenvironment (which has lowpH and is hypoxic).

Neutral Liposomes (NL) are Non-Cationic Lipid Based Particles.

Polymer nanoparticles are self-assembling polymer-based particles.

Double-stranded RNA binding motifs (dsRBMs) are self-assembling RNAbinding proteins, which will need modifications.

EPAS1 RNAi Agent Compositions in a Lipid Nanoparticles (LNP) Comprisinga Neutral Lipid; a Cationic Lipid; Cholesterol; and PEG-Lipid

Lipid nanoparticles (LNP) are self-assembling cationic lipid basedsystems. These can comprise, for example, a neutral lipid (the liposomebase); a cationic lipid (for siRNA loading); cholesterol (forstabilizing the liposomes); and PEG-lipid (for stabilizing theformulation, charge shielding and extended circulation in thebloodstream).

A Neutral Lipid

A neutral lipid is, for example, the liposome base.

A Cationic Lipid

A cationic lipid is, for example, for siRNA loading.

Cholesterol

Cholesterol is, for example, for stabilizing the liposomes)

PEG-lipid (for stabilizing the formulation, charge shielding andextended circulation in the bloodstream).

PEG-lipid is, for example, for stabilizing the formulation, chargeshielding and extended circulation in the bloodstream.

A Particular Formulation, and Ratios of a Neutral Lipid; a CationicLipid; Cholesterol; and PEG-Lipid.

In one aspect, the formulation comprises:

In one aspect, the formulation comprises:

In one aspect, the formulation comprises:

In one aspect, the formulation comprises:

PEG-Lipid

In one aspect, the formulation comprises a RNAi agent comprising anysequence disclosed herein, or any portion thereof (e.g., 15 or morecontiguous nt with 0, 1, 2 or 3 mismatches), optionally with anylengths, modifications, terminal dinucleotides, endcaps, combinations ofRNAi agents, combination therapy involving a EPAS1 RNAi agent andanother agent, conjugation with other components, compositions ormethods or techniques for delivery, and/or disease treatment (any ofwhich can be described herein or known in the art), further comprisingone or more of compound 1, cholesterol, DSPC, and/or PEG-lipid. In oneaspect, the formulation is 45% compound 1 (cationic lipid), 44%(cholesterol), 9% (DSPC) and 2% (PEG-lipid)—all molar ratios. These areknown in the art and/or described in U.S. Patent App. No. 61/774,759,filed Mar. 8, 2013. In one aspect, the disclosure pertains to acomposition comprising 45% compound 1 (cationic lipid), 44%(cholesterol), 9% (DSPC) and 2% (PEG-lipid) [all molar ratios] and aRNAi agent of the sequence of RNAi agent 5049. In one aspect, thedisclosure pertains to a composition comprising 45% compound 1 (cationiclipid), 44% (cholesterol), 9% (DSPC) and 2% (PEG-lipid) [all molarratios] and a RNAi agent of the sequence of RNAi agent 3875. In oneaspect, the disclosure pertains to a composition comprising 45% compound1 (cationic lipid), 44% (cholesterol), 9% (DSPC) and 2% (PEG-lipid) [allmolar ratios] and a RNAi agent comprising a first strand and a secondstrand, wherein the sequence of the first strand is or comprises thesequence of SEQ ID NO: 300, and/or the sequence of the second strand isor comprises the sequence of SEQ ID NO: 301.

In one aspect, the disclosure pertains to a composition comprising 45%compound 1 (cationic lipid), 44% (cholesterol), 9% (DSPC) and 2%(PEG-lipid) [all molar ratios] and a RNAi agent comprising a firststrand and a second strand, wherein the sequence of the first strand isor comprises the sequence of SEQ ID NO: 302, and/or the sequence of thesecond strand is or comprises the sequence of SEQ ID NO: 303. In oneaspect, the disclosure pertains to a composition comprising 45% compound1 (cationic lipid), 44% (cholesterol), 9% (DSPC) and 2% (PEG-lipid) [allmolar ratios] and a RNAi agent comprising a first strand and a secondstrand, wherein the sequence of the first strand is or comprises thesequence of SEQ ID NO: 303, and/or the sequence of the second strand isor comprises the sequence of SEQ ID NO: 304.

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:303 or nt 1-19 of SEQ ID NO: 303, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 304 or nt 1-19 of SEQID NO: 304, wherein the first and/or second strand are modified or notmodified.

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:303 or nt 1-19 of SEQ ID NO: 303, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 304 or nt 1-19 of SEQID NO: 304, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about10% cationic lipid [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:303 or nt 1-19 of SEQ ID NO: 303, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 304 or nt 1-19 of SEQID NO: 304, wherein the first and or second strand are modified or notmodified, and wherein the composition further comprises at least about20% cationic lipid [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:303 or nt 1-19 of SEQ ID NO: 303, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 304 or nt 1-19 of SEQID NO: 304, wherein the first and or second strand are modified or notmodified, and wherein the composition further comprises at least about30% cationic lipid [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:303 or nt 1-19 of SEQ ID NO: 303, and or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 304 or nt 1-19 of SEQID NO: 304, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about40% cationic lipid [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:303 or nt 1-19 of SEQ ID NO: 303, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 304 or nt 1-19 of SEQID NO: 304, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about45% cationic-lipid [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:303 or nt 1-19 of SEQ ID NO: 303, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 304 or nt 1-19 of SEQID NO. 304, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about10% compound 1 (cationic lipid) [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:303 or nt 1-19 of SEQ ID NO: 303, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 304 or nt 1-19 of SEQID NO: 304, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about20% compound 1 (cationic lipid) [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:303 or nt 1-19 of SEQ ID NO: 303, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 304 or nt 1-19 of SEQID NO: 304, wherein the first and for second strand are modified or notmodified, and wherein the composition further comprises at least about30% compound 1 (cationic lipid)/[molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:303 or nt 1-19 of SEQ ID NO: 303, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 304 or nt 1-19 of SEQID NO. 304, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about40% compound 1 (cationic lipid) [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:303 or nt 1-19 of SEQ ID NO: 303, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 304 or nt 1-19 of SEQID NO: 304, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about45% compound 1 (cationic lipid) [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:303 or nt 1-19 of SEQ ID NO: 303, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 304 or nt 1-19 of SEQit NO: 304, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about10% cholesterol [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:303 or nt 1-19 of SEQ ID NO: 303, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 304 or nt 1-19 of SEQID NO: 304, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about20% cholesterol [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi, agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:303 or nt 1-19 of SEQ ID NO: 303, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 304 or nt 1-19 of SEQID NO: 304, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about30% cholesterol [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:303 or nt 1-19 of SEQ ID NO: 303, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 304 or nt 1-19 of SEQID NO: 304, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about40% cholesterol [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ No: 303or nt 1-19 of SEQ ID NO: 303, and/or the sequence of the second strandis or comprises the sequence of SEQ ID NO: 304 or nt 1-19 of SEQ ID NO:304, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about44% cholesterol [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:303 or nt 1-19 of SEQ ID NO: 303, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 304 or nt 1-19 of SEQID NO: 304, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about1% DSPC [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:303 or nt 1-19 of SEQ ID NO: 303, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 304 or nt 1-19 of SEQID NO: 304, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about2% DSPC [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:303 or nt 1-19 of SEQ ID NO: 303, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 304 or nt 1-19 of SEQID NO: 304, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises al least about5% DSPC [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:303 or nt 1-19 of SEQ ID NO: 303, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 304 or nt 1-19 of SEQID NO: 304, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about7.5% DSPC [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:303 or nt 1-19 of SEQ ID NO: 303, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 304 of nt 1-19 of SEQID NO: 304, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about9% DSPC [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:303 or nt 1-19 of SEQ ID NO: 303, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 304 or nt 1-19 of SEQID NO: 304, wherein the first and or second strand are modified or notmodified, and wherein the composition further comprises at least about1% PEG [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:303 or nt 1-19 of SEQ ID NO: 303, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 304 or nt 1-19 of SEQID NO: 304, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about2% PEG [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:303 or nt 1-19 of SEQ ID NO: 303, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 304 or nt 1-19 of SEQID NO: 304, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about1% cDSA-PEG [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:303 or nt 1-19 of SEQ ID NO: 303, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 304 or nt 1-19 of SEQID NO: 304, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about2% cDSA-PEG [molar ratios].

Any of the recited amounts of components (compound 1, cationic lipid,cholesterol, DSPC, PEG, etc.) can be mixed/matched or combined, providedthey are not mutually exclusive, in various other aspects of thedisclosure.

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:300 or nt 1-19 of SEQ ID NO: 300, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 301 or nt 1-19 of SEQID NO: 301, wherein the first and/or second strand are modified or notmodified.

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:300 or nt 1-19 of SEQ ID NO: 300, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 301 or nt 1-19 of SEQID NO: 301, wherein the first and or second strand are modified or notmodified, and wherein the composition further comprises at least about10% cationic lipid [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:300 or nt 1-19 of SEQ ID NO: 300, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 301 or nt 1-19 of SEQID NO: 301, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about20% cationic lipid [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:300 or nt 1-19 of SEQ ID NO: 300, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 301 or nt 1-19 of SEQID NO: 301, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about30% cationic lipid [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:300 or nt 1-19 of SEQ ID NO: 300, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 301 or nt 1-19 of SEQID NO: 301, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about40% cationic lipid [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:300 or nt 1-19 of SEQ ID NO: 300, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 301 or nt 1-19 of SEQID NO: 301, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about45% cationic lipid [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:300 or nt 1-19 of SEQ ID NO: 300, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 301 or nt 1-19 of SEQID NO: 301, wherein the first and or second strand are modified or notmodified, and wherein the composition further comprises at least about10% compound 1 (cationic lipid) [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:300 or nt 1-19 of SEQ ID NO: 300, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 301 or nt 1-19 of SEQID NO: 301, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about20% compound 1 (cationic lipid) [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:300 or nt 1-19 of SEQ ID NO: 300, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 301 or nt 1-19 of SEQID NO: 301, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about30% compound 1 (cationic lipid) [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:300 or nt 1-19 of SEQ ID NO: 300, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 301 or nt 1-19 of SEQID NO: 301, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises al least about40% compound 1 (cationic lipid) [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:300 or nt 1-19 of SEQ ID NO: 300, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 301 or nt 1-19 of SEQID NO: 301, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about45% compound 1 (cationic lipid) [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:300 or nt 1-19 of SEQ ID NO: 300, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 301 or nt 1-19 of SEQID NO: 301, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about10% cholesterol [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:300 or nt 1-19 of SEQ ID NO: 300, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 301 or nt 1-19 of SEQID NO: 301, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about20% cholesterol [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:300 or nt 1-19 of SEQ ID NO: 300, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 301 or nt 1-19 of SEQID NO: 301, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about30% cholesterol [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:300 or nt 1-19 of SEQ ID NO: 300, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 301 or nt 1-19 of SEQID NO: 301, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about40% cholesterol [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:300 or nt 1-19 of SEQ ID NO: 300, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 301 or nt 1-19 of SEQID NO: 301, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about44% cholesterol [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:300 or nt 1-19 of SEQ ID NO: 300, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 301 or nt 1-19 of SEQID NO: 301, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about1% DSPC [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:300 or nt 1-19 of SEQ ID NO: 300, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 301 or nt 1-19 of SEQID NO: 301, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about2% DSPC [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:300 or nt 1-19 of SEQ ID NO: 300, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 301 or nt 1-19 of SEQID NO: 301, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about5% DSPC [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:300 or nt 1-19 of SEQ ID NO: 300, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 301 or nt 1-19 of SEQID NO: 301, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about7.5% DSPC [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:300 or nt 1-19 of SEQ ID NO: 300, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 301 or nt 1-19 of SEQID NO: 301, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about9% DSPC [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:300 or nt 1-19 of SEQ ID NO: 300, and or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 301 or nt 1-19 of SEQID NO: 301, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about1% PEG [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:300 or nt 1-19 of SEQ ID NO: 300, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 301 or nt 1-19 of SEQID NO: 301, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about2% PEG [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:300 or nt 1-19 of SEQ ID NO: 300, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 301 or nt 1-19 of SEQID NO: 301, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about1% cDSA-PEG [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:300 or nt 1-19 of SEQ ID NO: 300, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 301 or nt 1-19 of SEQID NO: 301, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about2% cDSA-PEG [molar ratios].

Any of the recited amounts of components (compound 1, cationic lipid,cholesterol, DSPC, PEG, etc.) can be mixed/matched or combined, providedthey are not mutually exclusive, in various other aspects of thedisclosure.

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:302 or nt 1-19 of SEQ ID NO: 302, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 305 or nt 1-19 of SEQID NO: 305, wherein the first and/or second strand are modified or notmodified.

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:302 or nt 1-19 of SEQ ID NO: 302, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 305 or nt 1-19 of SEQID NO: 305, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about10% compound 1 (cationic lipid) [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:302 or nt 1-19 of SEQ ID NO: 302, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 305 or nt 1-19 of SEQID NO: 305, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about20% compound 1 (cationic lipid) [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:302 or nt 1-19 of SEQ ID NO: 302, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 305 or nt 1-19 of SEQID NO: 305, wherein the first and or second strand are modified or notmodified, and wherein the composition further comprises at least about30% compound 1 (cationic lipid) [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:302 or nt 1-19 of SEQ ID NO: 302, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 305 or nt 1-19 of SEQID NO: 305, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about40% compound 1 (cationic lipid) [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:302 or nt 1-19 of SEQ ID NO: 302, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 305 or nt 1-19 of SEQID NO: 305, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about45% compound 1 (cationic lipid) [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:302 or nt 1-19 of SEQ ID NO: 302, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 305 or nt 1-19 of SEQID NO: 305, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about10% cationic lipid [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:302 or nt 1-19 of SEQ ID NO: 302, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 305 or nt 1-19 of SEQID NO: 305, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about20% cationic lipid [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:302 or nt 1-19 of SEQ ID NO: 302, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 305 or nt 1-19 of SEQID NO: 305, wherein the first and or second strand are modified or notmodified, and wherein the composition further comprises at least about30% cationic lipid [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:302 or nt 1-19 of SEQ ID NO: 302, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 305 or nt 1-19 of SEQID NO: 305, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about40% cationic lipid [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:302 or nt 1-19 of SEQ ID NO: 302, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 305 or nt 1-19 of SEQID NO: 305, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about45% cationic lipid [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:302 or nt 1-19 of SEQ ID NO: 302, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 305 or nt 1-19 of SEQID NO: 305, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about10% cholesterol [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:302 or nt 1-19 of SEQ ID NO: 302, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 305 or nt 1-19 of SEQID NO: 305, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about20% cholesterol [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:302 or nt 1-19 of SEQ ID NO: 302, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 305 or nt 1-19 of SEQID NO: 305, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about30% cholesterol [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:302 or nt 1-19 of SEQ ID NO: 302, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 305 or nt 1-19 of SEQID NO: 305, wherein the first and or second strand are modified or notmodified, and wherein the composition further comprises at least about40% cholesterol [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:302 or nt 1-19 of SEQ ID NO: 302, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 305 or nt 1-19 of SEQID NO: 305, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about44% cholesterol [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:302 or nt 1-19 of SEQ ID NO: 302, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 305 or nt 1-19 of SEQID NO: 305, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about1% DSPC [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:302 or nt 1-19 of SEQ ID NO: 302, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 305 or nt 1-19 of SEQID NO: 305, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about2% DSPC [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:302 or nt 1-19 of SEQ ID NO: 302, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 305 or nt 1-19 of SEQID NO: 305, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about5% DSPC [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:302 or nt 1-19 of SEQ ID NO: 302, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 305 or nt 1-19 of SEQID NO: 305, wherein the first and or second strand are modified or notmodified, and wherein the composition further comprises at least about7.5% DSPC [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:302 or nt 1-19 of SEQ ID NO: 302, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 305 or nt 1-19 of SEQID NO: 305, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about9% DSPC [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:302 or nt 1-19 of SEQ ID NO: 302, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 305 or nt 1-19 of SEQID NO: 305, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about1% PEG [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:302, or nt 1-19 of SEQ ID NO: 302, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 305 or nt 1-19 of SEQID NO: 305, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about2% PEG [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:302 or nt 1-19 of SEQ ID NO: 302, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 305 or nt 1-19 of SEQID NO: 305, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about1% cDSA-PEG [molar ratios].

In one aspect, the disclosure pertains to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thesequence of the first strand is or comprises the sequence of SEQ ID NO:302 or nt 1-19 of SEQ ID NO: 302, and/or the sequence of the secondstrand is or comprises the sequence of SEQ ID NO: 305 or nt 1-19 of SEQID NO: 305, wherein the first and/or second strand are modified or notmodified, and wherein the composition further comprises at least about2% cDSA-PEG [molar ratios].

Any of the recited amounts of components (compound 1, cationic lipid,cholesterol DSPC, PEG, etc.) can be mixed/matched or combined, providedthey are not mutually exclusive, in various other aspects of thedisclosure.

Additional Pharmaceutical Compositions

In various aspects, the RNAi agent to EPAS1 is packaged as a monotherapyinto a delivery vehicle, or may be further ligated to one or morediagnostic compound, reporter group, cross-linking agent,nuclease-resistance conferring moiety, natural or unusual nucleobase,lipophilic molecule, cholesterol, lipid, lectin, steroid, uvaol,hecigenin, diosgenin, terpene, triterpene, sarsasapogenin, Friedelin,epifriedelanol-derivatized lithocholic acid, vitamin, carbohydrate,dextran, pullulan, chitin, chitosan, synthetic carbohydrate, oligolactate 15-mer, natural polymer, low- or medium-molecular weightpolymer, inulin, cyclodextrin, hyaluronic acid, protein, protein-bindingagent, integrin-targeting molecule, polycationic, peptide, polyamine,peptide mimic, and/or transferrin.

The RNAi agents of the present disclosure can be prepared in apharmaceutical composition comprising various components appropriate forthe particular method of administration of the RNAi agent.

Particular Specific Aspects

In a particular specific aspect, the present disclosure is a compositioncomprising one or more EPAS1 RNAi agents.

In various aspects, the disclosure encompasses a composition comprisingany one or more of the following:

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 39, and thesequence of the second strand is the sequence of SEQ ID NO: 58, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 40, and thesequence of the second strand is the sequence of SEQ ID NO: 59, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 41, and thesequence of the second strand is the sequence of SEQ ID NO: 60, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 42, and thesequence of the second strand is the sequence of SEQ ID NO: 61, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 43, and thesequence of the second strand is the sequence of SEQ ID NO: 62, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 44, and thesequence of the second strand is the sequence of SEQ ID NO: 63, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the fast strand is the sequence of SEQ ID NO: 45, and thesequence of the second strand is the sequence of SEQ ID NO: 64, ormodified, or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 46, and thesequence of the second strand is the sequence of SEQ ID NO: 65, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 47, and thesequence of the second strand is the sequence of SEQ ID NO: 66, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence, of the first strand is the sequence of SEQ ID NO: 48, and thesequence of the second strand is the sequence of SEQ ID NO: 67, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 48, and thesequence of the second strand is the sequence of SEQ ID NO: 67, whereinthe sequence of the first and/or second strand further comprises a 3′terminal dinucleotide, or modified or unmodified variants of the RNAiagent.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 48, and thesequence of the second strand is the sequence of SEQ ID NO: 67, whereinthe sequence of the first and/or second strand further comprises a 3′terminal dinucleotide selected from TT, UU, U (2′-OMe) dT, U (2′-OMe)(2′-OMe), T(2′-OMe) T (2′-OMe), T(2′-OMe) dT, dTdT, sdT, dTsdT, sdTsdT,and sdTdT, or modified or unmodified variants of the RNAi agent.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 48, and thesequence of the second strand is the sequence of SEQ ID NO: 67, whereinthe sequence of the first and/or second strand further comprises a 3′ UUterminal dinucleotide, or modified or unmodified variants of the RNAiagent.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 49, and thesequence of the second strand is the sequence of SEQ ID NO: 68, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 50, and thesequence of the second strand is the sequence of SEQ ID NO: 69, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 51, and thesequence of the second strand is the sequence of SEQ ID NO: 70, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 51, and thesequence of the second strand is the sequence of SEQ ID NO: 70, whereinthe sequence of the first and/or second strand further comprises a 3′terminal dinucleotide, or modified or unmodified variants of the RNAiagent.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 51, and thesequence of the second strand is the sequence of SEQ ID NO: 70, whereinthe sequence of the first and/or second strand further comprises a 3′terminal dinucleotide selected from TT, UU, U (2′-OMe) dT, U (2′-OMe) U(2′-OMe), T(2′-OMe) T (2′-OMe), T(2′-OMe) dT, dTdT, sdT, dTsdT, sdTsdT,and sdTdT, or modified or unmodified variants of the RNAi agent.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 51, and thesequence of the second strand is the sequence of SEQ ID NO: 70, whereinthe sequence of the first and/or second strand further comprises a 3′ UUterminal dinucleotide, or modified or unmodified variants of the RNAiagent.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 52, and thesequence of the second strand is the sequence of SEQ ID NO: 71, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 53, and thesequence of the second strand is the sequence of SEQ ID NO: 72, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 54, and thesequence of the second strand is the sequence of SEQ ID NO: 73, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 55, and thesequence of the second strand is the sequence of SEQ ID NO: 74, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 56, and thesequence of the second strand is the sequence of SEQ ID NO: 75, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 57, and thesequence of the second strand is the sequence of SEQ ID NO: 76, ormodified or unmodified variants thereof.

When reference is made herein to a RNAi wherein the sequence of onestrand “is” the sequence of a recited SEQ ID NO., the referenceindicates that the sequence of the one strand “consists of” the sequenceof the recited SEQ ID NO.

Additional Particular Specific Aspects

In a particular specific aspect, the present disclosure is a compositioncomprising one or more EPAS1 RNAi agents.

In various aspects, the disclosure encompasses a composition comprisingany one or more of the following:

Additional Particular Specific Aspects

In a particular specific aspect, the present disclosure is a compositioncomprising one or more EPAS1 RNAi agents.

In various aspects, the disclosure encompasses a composition comprisingany one or more of the following:

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 39, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 40, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 41, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 42, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 43, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 44, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 45, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 46, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 47, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 48, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and second strand, wherein the sequenceof the first strand is the sequence of SEQ ID NO: 48, wherein thesequence of the strand further comprises a 3′ terminal dinucleotide, ormodified or unmodified variants of the RNAi agent.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 48, whereinthe sequence of the strand further comprises a 3′ terminal dinucleotideselected from TT, UU, U (2′-OMe) dT, U (2′-OMe) U (2′-OMe), T(2′-OMe) T(2′-OMe), T(2′-OMe) dT, dTdT, sdT, dTsdT, sdTsdT, and sdTdT, or modifiedor unmodified variants of the RNAi agent.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 48, whereinthe sequence of the strand further comprises a 3′ terminal UUdinucleotide, or modified, or unmodified variants of the RNAi agent.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 49, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 50, ormodified or unmodified variants thereof.

A RNAi of comprising a first and a second strand, wherein the sequenceof the first strand is the sequence of SEQ ID NO: 51 or modified orunmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 51, whereinthe sequence of the first strand further comprises a 3′ terminaldinucleotide, or modified or unmodified variants of the RNAi agent.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 51, whereinthe sequence of the strand farther comprises a 3′ terminal dinucleotideselected from TT, UU, U (2′-OMe) dT, U (2′-OMe) U (2′-OMe), T(2′-OMe) T(2′-OMe), T(2′-OMe) dT, sdT, dTsdr, sdTsdT, and sdTdT, or modified orunmodified variants of the RNAi agent.

A RNAi agent comprising a first and a second, strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 51, whereinthe sequence of the strand further comprises a 3′ UU terminaldinucleotide, or modified or unmodified variants of the RNAi agent.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 52, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 53, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand, is the sequence of SEQ ID NO: 54, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 55, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 56, ormodified or unmodified variants thereof.

A RNAi agent comprising it first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 57, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 58, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 59, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 60, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 61, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 62, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 63, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 64, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 65, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 66, ormodified or unmodified variants thereof.

A RNAi argent comprising a first and second strand, wherein the sequenceof the first strand is the sequence of SEQ ID NO: 67, or modified orunmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 67, whereinthe sequence of the strand further comprises a 3′ terminal dinucleotide,or modified or unmodified variants of the RNAi agent.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 67, whereinthe sequence of the strand further comprises a 3′ terminal dinucleotideselected from TT, UU, U (2′-OMe) dT, U (2′-OMe) U (2′-OMe), T (2′-OMe),T(2′-OMe) dT, dTdT, sdT, sdTsdT, and sdTdT, or modified or unmodifiedvariants of the RNAi agent.

A RNAi comprising a first and a second strand, wherein the sequence ofthe first strand is the sequence of SEQ ID NO: 67, wherein the sequenceof the strand further comprises a 3′ terminal UU dinucleotide, ormodified or unmodified variants of the RNAi agent.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 68, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 69, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 70, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 70, whereinthe sequence of the strand further comprises a 3′ terminal dinucleotide,or modified or unmodified variants of the RNAi agent.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 70, whereinthe sequence of the strand farther comprises a 3′ terminal dinucleotideselected from TT, UU, U (2′-OMe) dT, U (2′-OMe) U (2′-OMe), T(2′-OMe) T(2′-OMe), T(2′-OMe) dT, dTdT, sdT, dTsdT, sdTsdT, and sdTdT, or modifiedor unmodified variants of the RNAi agent.

A RNAi agent comprising a first and a second, strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 70, whereinthe sequence of the strand further comprises a 3′ terminal UUdinucleotide, or modified or unmodified variants of the RNAi agent.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 71, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 72, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 73, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 74, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 75, ormodified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand is the sequence of SEQ ID NO: 76, ormodified or unmodified variants thereof.

When reference is made herein to a RNAi wherein the sequence of onestrand “is” the sequence of a recited SEQ ID NO., the referenceindicates that the sequence of the one strand consists or the sequenceof the recited SEQ ID NO.

Additional Particular Specific Aspects

In various aspects, the disclosure comprises a RNAi agent comprising afirst and a second strand, wherein the sequence of the first strandcomprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3nt from a first strand, and/or the sequence of the second strandcomprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3nt from the second strand of any one or more RNAi agent disclosedherein.

In various aspects, the disclosure encompasses a composition comprisingany one or more of the following:

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nt from the sequence of SEQ IDNO: 39, and/or the sequence of the second strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sequenceof SEQ ID NO. 58, wherein the length of the first and second strand areeach no more than about 30 nt, or modified or unmodified variantsthereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nt from the sequence of SEQ IDNO: 40, and/or the sequence of the second strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sequenceof SEQ ID NO: 59, wherein the length of the first and second strand areeach no more than about 30 nt, or modified or unmodified variantsthereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nt from the sequence of SEQ IDNO: 41, and/or the sequence of the second strand comprises at leastcontiguous nucleotides differing by 0, 1, 2, or 3 at from the sequenceof SEQ ID NO: 60, wherein the length of the first and second strand areeach no more than about 30 nt, or modified or unmodified variantsthereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2 or 3 nt from the sequence of SEQ ID NO:42, and/or the sequence of the second strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sequenceof SEQ ID NO: 61, wherein the length of the first and second strand areeach no more than about 30 nt, or modified or unmodified variantsthereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nt from the sequence of ID NO:43, and/or the sequence of the second strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sequenceof SEQ ID NO: 62, wherein the length of the first and second strand areeach no more than about 30 nt, or modified or unmodified variantsthereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nt from the sequence of SEQ IDNO: 44, and/or the sequence of the second strand comprises at least 15contiguous nucleotides, differing by 0, 1, 2, or 3 n t from the sequenceof SEQ ID NO: 63, wherein the length of the first and second strand areeach no more than about 30 nt, or modified or unmodified variantsthereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nt from the sequence of SEQ IDNO: 45, and/or the sequence of the second strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sequenceof SEQ ID NO: 64, wherein the length of the first and second strand areeach no more than about 30 nt, or modified or unmodified variantsthereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nt from the sequence of SEQ IDNO: 46, and/or the sequence of the second strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sequenceof SEQ ID NO: 65, wherein the length of the first and second strand areeach no more than about 30 nt, or modified or unmodified variantsthereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2 or 3 nt from the sequence of SEQ ID NO:47, and/or the sequence of the second strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sequenceof SEQ ID NO: 66, wherein the length of the first and second strand areeach no more than about 30 nt, or modified or unmodified variantsthereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nt from the sequence of ID NO:48, and/or the sequence of the second strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sequenceof SEQ ID NO: 67, wherein the length of the first and second strand areeach no more than about 30 nt, or modified or unmodified variantsthereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nt from the sequence of SEQ IDNO: 49, and/or the sequence of the second strand comprises at least 15contiguous nucleotides, differing by 0, 1, 2, or 3 nt from the sequenceof SEQ ID NO: 68, wherein the length of the first and second strand areeach no more than about 30 nt, or modified or unmodified variantsthereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nt from the sequence of SEQ IDNO: 50, and/or the sequence of the second strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sequenceof SEQ ID NO: 69, wherein the length of the first and second strand areeach no more than about 30 nt, or modified or unmodified variantsthereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nt from the sequence of SEQ IDNO: 51, and/or the sequence of the second strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sequenceof SEQ ID NO: 70, wherein the length of the first and second strand areeach no more than about 30 nt, or modified or unmodified variantsthereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2 or 3 nt from the sequence of SEQ ID NO:52, and/or the sequence of the second strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sequenceof SEQ ID NO: 71, wherein the length of the first and second strand areeach no more than about 30 nt, or modified or unmodified variantsthereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nt from the sequence of SEQ IDNO: 53, and/or the sequence of the second strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sequenceof SEQ ID NO: 72, wherein the length of the first and second strand areeach no more than about 30 nt, or modified or unmodified variantsthereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nt from the sequence of SEQ IDNO: 54, and/or the sequence of the second strand comprises at least 15contiguous nucleotides, differing by 0, 1, 2, or 3 nt from the sequenceof SEQ ID NO: 73, wherein the length of the first and second strand areeach no more than about 30 nt, or modified or unmodified variantsthereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nt from the sequence of SEQ IDNO: 55, and/or the sequence of the second strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sequenceof SEQ ID NO: 74, wherein the length of the first and second strand areeach no more than about 30 nt, or modified or unmodified variantsthereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nt from the sequence of SEQ IDNO: 50, and/or the sequence of the second strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 in from the sequenceof SEQ ID NO: 75, wherein the length of the first and second strand areeach no more than about 30 nt, or modified or unmodified variantsthereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2 or 3 nt from the sequence of SEQ ID NO:57, and/or the sequence of the second strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sequenceof SEQ ID NO: 76, wherein the length of the first and second strand areeach no more than about 30 nt, or modified or unmodified variantsthereof.

Additional Particular Specific Aspects

In various aspects, the disclosure comprises a RNAi agent comprising afirst and a second strand, wherein the sequence of the first strandcomprises at least 15 contiguous nucleotides of a first strand, and/orthe sequence of the second strand comprises at least 15 contiguousnucleotides of the second strand of any one or more RNAi agent disclosedherein.

In various aspects, the disclosure encompasses a composition comprisingany one or more of the following:

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises at least 15 contiguousnucleotides of the sequence of SEQ ID NO: 39, and/or the sequence of thesecond strand comprises at least 15 contiguous nucleotides of thesequence of SEQ ID NO: 58, wherein the length of the first and secondstrand are each no more than about 30 nt, or modified or unmodifiedvariants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises at least 15 contiguousnucleotides of the sequence of SEQ ID NO: 40, and/or the sequence of thesecond strand comprises at least 15 contiguous nucleotides of thesequence of SEQ ID NO: 59, wherein the length of the first and secondstrand are each no more than about 30 nt, or modified or unmodifiedvariants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises at least 15 contiguousnucleotides of the sequence of SEQ ID NO: 41, and/or the sequence of thesecond strand comprises at least 15 contiguous nucleotides of thesequence of SEQ ID NO: 60, wherein the length of the first and secondstrand are each no more than about 30 nt, or modified or unmodifiedvariants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises at least 15 contiguousnucleotides of the sequence of SEQ ID NO: 42, and/or the sequence of thesecond strand comprises at least 15 contiguous nucleotides of thesequence of SEQ ID NO: 61, wherein the length of the first and secondstrand are each no more than about 30 nt, or modified or unmodifiedvariants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises at least 15 contiguousnucleotides of the sequence of SEQ ID NO: 43, and/or the sequence of thesecond strand comprises at least 15 contiguous nucleotides of theSequence of SEQ ID NO: 62, wherein the length of the first and secondstrand are each no more than about 30 nt, or modified or unmodifiedvariants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises at least 15 contiguousnucleotides of the Sequence of SEQ ID NO: 44, and/or the sequence of thesecond strand comprises at least 15 contiguous nucleotides of thesequence of SEQ ID NO: 63, wherein the length of the first and secondstrand are each no more than about 30 nt, or modified or unmodifiedvariants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises at least 15 contiguousnucleotides of the sequence of SEQ ID NO: 45, and/or the sequence of thesecond strand comprises at least 15 contiguous nucleotides of thesequence of SEQ ID NO: 64, wherein the length of the first and secondstrand are each no more than about 30 nt, or modified or unmodifiedvariants thereof.

A RNAi agent comprising a first and a second, strand wherein thesequence of the first strand comprises at least 15 contiguousnucleotides of the sequence of SEQ ID NO: 46, and/or the sequence of thesecond strand comprises at least 15 contiguous nucleotides of thesequence of SEQ ID NO: 65, wherein the length of the first and secondstrand are each no more than about 30 nt, or modified or unmodifiedvariants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first, strand comprises at least 15 contiguousnucleotides of the sequence of SEQ ID NO: 47, and/or the sequence of thesecond strand comprises at least 15 contiguous nucleotides of thesequence of SEQ ID NO: 66, wherein the length of the first and secondstrand are each no more than about 30 nt, or modified or unmodifiedvariants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises at least 15 contiguousnucleotides of the sequence of SEQ ID NO: 48, and/or the sequence of thesecond strand comprises at least 15 contiguous nucleotides of thesequence of SEQ ID NO: 67, wherein the length of the first and secondstrand are each no more than about 30 nt, or modified or unmodifiedvariants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises at least 15 contiguousnucleotides of the sequence of SEQ ID NO: 49, and/or the sequence of thesecond strand comprises at least 15 contiguous nucleotides of thesequence of SEQ ID NO: 68, wherein the length of the first and secondstrand are each no more than about 30 nt, or modified unmodifiedvariants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises at least 15 contiguousnucleotides of the sequence of SEQ ID NO: 50, and/or the sequence of thesecond strand comprises at least 15 contiguous nucleotides of thesequence of SEQ ID NO: 69, wherein the length of the first and secondstrand are each no more than about 30 nt, or modified or unmodifiedvariants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises at least 15 contiguousnucleotides of the sequence of SEQ ID NO: 51, and/or the sequence of thesecond strand comprises at least 15 contiguous nucleotides of thesequence of SEQ ID NO: 70, wherein the length of the first and secondstrand are each no more than about 30 nt, or modified or unmodifiedvariants thereof.

A RNAi agent comprising a first and a second, strand, wherein thesequence of the first strand comprises at least 15 contiguousnucleotides of the sequence of SEQ ID NO: 52, and/or the sequence of thesecond strand comprises at least 15 contiguous nucleotides of thesequence of SEQ ID NO: 71, wherein the length of the first and secondstrand are each no more than about 30 nt, or modified or unmodifiedvariants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises at least 15 contiguousnucleotides of the sequence of SEQ ID NO: 53, and/or the sequence of thesecond strand comprises at least 15 contiguous nucleotides of thesequence of SEQ ID NO: 72, wherein the length of the first and secondstrand are each no more than about 30 nt, or modified or unmodifiedvariants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises at least 15 contiguousnucleotides of the sequence of SEQ ID NO: 54, and/or the sequence of thesecond strand comprises at least 15 contiguous nucleotides of thesequence of SEQ ID NO; 73, wherein the length of the first and secondstrand are each no more than about 30 nt, or modified or unmodifiedvariants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises at least 15 contiguousnucleotides of the sequence of SEQ ID NO: 55, and/or the sequence of thesecond strand comprises at least 15 contiguous nucleotides of thesequence of SEQ ID NO: 74, wherein the length of the first and secondstrand are each no more than about 30 nt, or modified or unmodifiedvariants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises at least 15 contiguousnucleotides of the sequence of SEQ ID NO: 56, and/or the sequence of thesecond strand comprises at least 15 contiguous nucleotides of thesequence of SEQ ID NO: 75, wherein the length of the first and secondstrand are each no more than about 30 nt, or modified or unmodifiedvariants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises at least 15 contiguousnucleotides of the sequence of SEQ ID NO: 57, and/or the sequence of thesecond strand comprises at least 15 contiguous nucleotides of thesequence of SEQ ID NO: 76, wherein the length of the first and secondstrand are each no more than about 30 nt or modified or unmodifiedvariants thereof.

Additional Particular Specific Aspects

In a particular specific aspect, the present disclosure is a compositioncomprising one or more EPAS1 RNAi agents.

In various aspects, the disclosure encompasses a composition comprisingany one or more of the following:

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 39,and/or the sequence of the second strand comprises the sequence of SEQID NO: 58, wherein the length of the first and second strand are each nomore than about 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 40,and/or the sequence of the second strand comprises the sequence of SEQID NO: 59, wherein the length of the first and second strand are each nomore than about 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 41,and/or the sequence of the second strand comprises the sequence of SEQID NO: 60, wherein the length of the first and second strand are each nomore than about 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 42,and/or the sequence of the second strand comprises the sequence of SEQID NO: 61, wherein the length of the first and second strand are each nomore than about 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 43,and/or the sequence of the second strand comprises the sequence of SEQID NO: 62, wherein the length of the first and second strand are each nomore than about 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 44,and/or the sequence of the second strand comprises the sequence of SEQID NO: 63, wherein the length of the first and second strand are each nomore than about 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 45,and/or the sequence of the second strand comprises the sequence of SEQID NO: 64, wherein the length of the first and second strand are each nomore than about 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 46,and/or the sequence of the second strand comprises the sequence of SEQID NO: 65, wherein the length of the first and second strand are each nomore than about 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 47,and/or the sequence of the second strand comprises the sequence of SEQID NO: 66, wherein the length of the first and second strand are each nomore than about 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 48,and/or the sequence of the second strand comprises the sequence of SEQID NO: 67, wherein the length of the first and second strand are each nomore than about 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 49,and/or the sequence of the second strand comprises the sequence of SEQID NO: 68, wherein the length of the first and second strand are each nomore than about 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 50,and/or the sequence of the second strand comprises the sequence SEQ IDNO: 69, wherein the length of the first and second strand are each nomore than about 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 51,and/or the sequence of the second strand comprises the sequence of SEQID NO: 70, wherein the length of the first and second strand are each nomore than about 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 52,and/or the sequence of the second strand comprises the sequence of SEQID NO; 71, wherein the length of the first and second strand are each nomore than about 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 53,and/or the sequence of the second strand comprises the sequence of SEQID NO: 72, wherein the length of the first and second strand are each nomore than about 30 nt or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 54,and/or the sequence of the second strand comprises the sequence of SEQID NO: 73, wherein the length of the first and second strand are each nomore than about 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 55,and/or the sequence of the second strand comprises the sequence of SEQID NO: 74, wherein the length of the first and second strand are each nomore than about 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 56,and/or the sequence of the second strand comprises the sequence of SEQID NO; 75, wherein the length of the first and second strand are each nomore than about 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 57,and/or the sequence of the second strand comprises the sequence of SEQID NO: 76, wherein the length of the first and second strand are each nomore than about 30 nt, or modified or unmodified variants thereof.

Additional Particular Specific Aspects

In a particular specific aspect, the present disclosure is a compositioncomprising one or more EPAS1 RNAi agents.

In various aspects, the disclosure encompasses a composition comprisingany one or more of the following:

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 39,wherein the length of the first and second strand are each no more thanabout 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 40,wherein the length of the first and second strand are each no more thanabout 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 41,wherein the length of the first and second strand are each no more thanabout 30 nt, or modified or unmodified variants thereof.

A RNAi comprising a first and a second strand, wherein the sequence ofthe first strand comprises the sequence of SEQ ID NO; 42, wherein thelength of the first and second strand are, each no more than about 30nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 43,wherein the length of the first and second strand are each no more thanabout 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 44,wherein the length of the first and second strand are each no more thanabout 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 45,wherein the length of the first and second strand are each no more thanabout 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 46,wherein the length of the first and second strand are each no more thanabout 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence, of the first strand comprises the sequence of SEQ ID NO: 47,wherein the length of the first and second strand are each no more thanabout 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 48,wherein the length of the first and second strand are each no more thanabout 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 49,wherein the length of the first and second strand are each no more thanabout 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence, of SEQ ID NO: 50,wherein the length of the first and second strand are each no more thanabout 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 51,wherein the length of the first and second strand are each no more thanabout 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 52,wherein the length of the first and second strand are each no more thanabout 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 53,wherein the length of the first and second strand are each no more thanabout 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 54,wherein the length of the first and second strand are each no more thanabout 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 55,wherein the length of the first and second strand are each no more thanabout 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 56,wherein the length of the first and second strand are each no more thanabout 30 nt or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 57,wherein the length of the first and second strand are each no more thanabout 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 58,wherein the length of the first and second strand are each no more thanabout 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 59,wherein the length of the first and second strand are each no more thanabout 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 60,wherein the length of the first and second strand are each no more thanabout 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 61,wherein the length of the first and second strand are each no more thanabout 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 62,wherein the length of the first and second strand are each no more thanabout 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 63,wherein the length of the first and second strand are each no more thanabout 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 64,wherein the length of the first and second strand are each no more thanabout 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 65,wherein the length of the first and second strand are each no more thanabout 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 66,wherein the length of the first and second strand are each no more thanabout 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 67,wherein the length of the first and second strand are each no more thanabout 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 68,wherein the length of the first and second strand are each no more thanabout 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 69,wherein the length of the first and second strand are each no more thanabout 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 70,wherein the length of the first and second strand are each no more thanabout 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 71,wherein the length of the first and second strand are each no more thanabout 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 72,wherein the length of the first and second strand are each no more thanabout 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 73,wherein the length of the first and second strand are each no more thanabout 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 74,wherein the length of the first and second strand are each no more thanabout 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 75,wherein the length of the first and second strand are each no more thanabout 30 nt, or modified or unmodified variants thereof.

A RNAi agent comprising a first and a second strand, wherein thesequence of the first strand comprises the sequence of SEQ ID NO: 76,wherein the length of the first and second strand are each no more thanabout 30 nt, or modified or unmodified variants thereof.

Additional Particular Aspects

In various aspects, the disclosure comprises a RNAi agent comprising asense and an antisense strand, wherein the antisense strand comprises atleast 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from theantisense strand of any RNAi agent disclosed herein, or modified orunmodified variants thereof, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nt (or anyrange thereof, e.g., 0-1, 1-2, 1-3, 1-4 nt, etc.).

Thus, in various aspects, the disclosure comprises a RNAi agentcomprising a sense and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3nt from the antisense strand of any of:

SEQ ID NOs: 40 and 59; SEQ ID NOs: 41 and 60; SEQ ID NOs: 42 and 61; SEQID NOs: 43 and 62; SEQ ID NOs: 44 and 63; SEQ ID NOs: 45 and 64; SEQ IDNOs: 46 and 65; SEQ ID NOs: 47 and 66; SEQ ID NOs: 48 and 67; SEQ IDNOs: 49 and 68; SEQ ID NOs: 50 and 69; SEQ ID NOs: 51 and 70; SEQ IDNOs: 52 and 71; SEQ ID NOs: 53 and 72; SEQ ID NOs: 54 and 73; SEQ IDNOs: 55 and 74; SEQ ID NOs: 56 and 75; or SEQ ID NOs: 57 and 76, ormodified or unmodified variants thereof, wherein the antisense strandoptionally further comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or morent (or any range thereof, e.g., 0-1, 1-2, 1-3, 1-4 nt, etc.).

In one aspect, the disclosure comprises any one or more RNAi agentlisted herein.

Additional Particular Specific Aspects

Other particular specific aspects include compositions comprising 1, 2,3, 4, or more of these RNAi agents. Another aspect is a compositioncomprising any single RNAi agent, along with any other RNAi agents whichoverlap it. Another aspect comprises two, three, four or more EPAS1 RNAiagents which do not overlap and thus target different parts of the RNAmolecule. When two or more RNAi agents are used, they can beadministered simultaneously or sequentially.

Another particular specific aspect comprises an RNAi agent, wherein theRNAi agent comprises a sense strand comprising at least 15 contiguousnucleotides (identical in sequence) to the sense strand of any of thelisted RNAi agents, and an antisense strand comprising at least 15contiguous nucleotides (identical in sequence) to the antisense strandof the same RNAi agent. In another aspect, the composition comprisesone, two, three, four, or more such RNAi agents.

In one aspect, the composition comprises an RNAi agent which comprisesan antisense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2 or 3 mismatches from the antisense strand of a RNAiagent described herein.

In one aspect, the composition comprises an RNAi agent which comprisesan antisense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2 or 3 mismatches from the antisense strand of a RNAiagent described herein.

In another aspect, the composition comprises an RNAi agent whichcomprises a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 mismatches from the sense strand of one ofthe listed RNAi agents, and an antisense strand comprising at least 15contiguous nucleotides differing by 0, 1, 2 or 3 mismatches from theantisense strand of the same RNAi agent.

A “mismatch” is defined herein as a difference between the base sequenceor length when two sequences are maximally aligned and compared. As anon-limiting example, a mismatch is counted if a difference existsbetween the base at a particular location in one sequence and the baseat the corresponding position in another sequence (e.g., between thesequence of a given RNAi agent and an RNAi agent listed herein). Thus, amismatch is counted, for example, if a position in one sequence has aparticular base (e.g. A), and the corresponding position on the othersequence has a different base (e.g., G, C or V). A mismatch is alsocounted, e.g., if a position in one sequence has a base (e.g., A), andthe corresponding position on the other sequence has no base (e.g., thatposition is an abasic nucleotide which comprises a phosphate-sugarbackbone but no base). A single stranded nick in either sequence (or inthe sense or antisense strand) is not counted as mismatch. Thus, as anon-limiting example, no mismatch would be counted if one sequencecomprises the sequence A-G, but the other sequence comprises thesequence A-G with a single-stranded nick between the A and the G. A basemodification is also not considered a mismatch, if one sequencecomprises a C, and the other sequence composes a modified C (e.g., witha 2′-modification) at the same position, no mismatch would be counted.Thus, modifications of a nucleotide other than replacement or alterationof the base would not constitute a mismatch. For example, no mismatchwould occur between a nucleotide which is A, and a nucleotide which is Awith a 5′ modification (e.g., those illustrated in FIG. 1) and/or a2′-modification. The key feature of a mismatch (base replacement) isthat it would not be able to base-pair with the corresponding base onthe opposite strand. In addition, terminal overhangs such as “UU” or“dTdT” are not counted when counting the number of mismatches; theterminal “UU” and “dTdT” overhangs are also not included whencalculating “15 contiguous nucleotides.”

In these aspects, a mismatch is defined as a position wherein the baseof one sequence does not match the base of the other sequence.

In another aspect, the composition comprises 1, 2, 3, 4, or more suchRNAi agents.

In another aspect, the composition comprises an RNAi agent whichcomprises a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2 or 3 mismatches from the sense strand of one of thelisted RNAi agents, and an antisense strand comprising at least 15contiguous nucleotides differing by 0, 1, 2 or 3 mismatches from theantisense strand of the same RNAi agent.

Overlapping Groups of EPAS1 siRNAs

In various aspects, the disclosure relates to groups of RNAi agents withoverlapping sequences. Thus, the disclosure encompasses groups of RNAiagents wherein each RNAi agent in the group overlaps with each otherRNAi agent in the same group by at least 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19 or more nucleotides. Particularly, in one aspect,the overlap is at least 12 nt. Groups of sequences that overlap areshown in Table 9 (Example 6), wherein each member of a group overlapswith each other member of the same group by at least 12 nt. A portion ofthe overlap of the sense strands and a portion of the overlap of theantisense strand are presented. Thus, for example, 3304 and 3310 sharethe common technical feature of the sequence of UCACUUUAUUAUC (SEQ IDNO: 115) in the sense strand, and the sequence of GAUAAUAAAGUGA (SEQ IDNO: 120) in the antisense strand. Note, of course, that variousgroupings comprise different numbers of overlapping siRNAs; any twosiRNAs within that group overlap. Thus, 5057, 5058 and 5059 all overlapwith each other, meaning that any two of that group (5057 and 5059; or5058 and 5059; or 5058 and 5059) share a common technical feature of anoverlapping portion of the sense and/or anti-sense strand sequence. Thedisclosure thus encompasses any pair or grouping of overlapping siRNAs,wherein the pair share a technical feature, namely, the portion of thesense and/or anti-sense strand which overlaps, as described in Table 9.

The disclosure thus encompasses various aspects comprising groups ofoverlapping RNAi agents, for example (1) RNAi agents comprising thesequences of 5057 and 5059; or 5058 and 5059; or 5058 and 5059 (or anyother pair or group of overlapping RNAi agents); (2) RNAi agentsconsisting of the sequences of 5057 and 5059; or 5058 and 5059; or 5058and 5059 (or any other pair or group of overlapping RNAi agents); (3)RNAi agents comprising the sequences of 5057 and 5059; or 5058 and 5059;or 5058 and 5059 (or any other pair or group of overlapping RNAiagents); (4) RNAi agents comprising a sense strand and/or a antisensestrand comprising a sequence of 5057 and 5059; or 5058 and 5059; or 5058and 5059 (or any other pair or group of overlapping RNAi agents); (5)RNAi agents comprising a sense strand and/or a antisense strandcomprising 15 contiguous nt with 0 to 3 mismatches from a sequence of5057 and 5059; or 5058 and 5059; or 5058 and 5059 (or any other pair orgroup of overlapping RNAi agents); (6) RNAi agents comprising a sensestrand comprising 15 contiguous nt with 0 to 3 mismatches from asequence of 5057 and 5059; or 5058 and 5059; or 5058 and 5059 (or anyother pair or group of overlapping RNAi agents); (7) RNAi agentscomprising an antisense strand comprising 15 contiguous nt with 0 to 3mismatches from a sequence of 5057 and 5059; or 5058 and 5059; or 5058and 5059 (or any other pair or group of overlapping RNAi agents); etc.The disclosure also encompasses similar aspects reflecting all theoverlapping groups of RNAi agents as described in Table 9.

Variants of RNAi agents (e.g., comprising different modifications, caps,etc.) are disclosed herein, e.g., in the Tables. An unmodified and anexample modified variant of various RNAi agents are provided. Thedisclosure thus encompasses groups of overlapping modified and/orunmodified RNAi agents. More aspects are provided herein, and areincluded in the scope of each RNAi agents of the disclosure.

EXAMPLES Example 1. Bioinformatics

Hundreds of RNAi agents (siRNAs) to EPAS1 were chosen based on severalcriteria. Some of the criteria (not all of which were met by allselected sequences) include: Human/Cyno cross reactivity; BioPred;siRNAs have U or A at nt 1 (although 2802 and 3739 start with a C);siRNAs exclude seed match to known human microRNA for AS strand; andsequences exclude repeats of 4 or more.

A selection of the most efficacious RNAi agents tested are disclosedherein, and their sequences presented in the Tables. Tables 2 and 3below present the DNA sequences of the RNAi agents, and the unmodifiedRNA sequence.

The phrase “Start” denotes the starting position of the oligonucleotide(e.g., the 19-mer) on the transcript. This is measured in nucleotidecoordinates, relative to beginning of the transcript.

TABLE 2 EPAS1 19-mers. DNA sequences. SEQ SEQ Position ID ID NM_001430Sense NO: Antisense NO:  842 ATGGCGACATGATCTTTCT  1 AGAAAGATCATGTCGCCAT20 2802 AAATGTACCCAATGATAAG  2 CTTATCATTGGGTACATTT 21 3040GAACTGACCAGATATGACT  3 AGTCATATCTGGTCAGTTC 22 3304 AGATGCTCACTTTATTATC 4 GATAATAAAGTGAGCATCT 23 3310 TCACTTTATTATCCCTATT  5AATAGGGATAATAAAGTGA 24 3345 GTTTTACCTGTTCTGAAAT  6 ATTTCAGAACAGGTAAAAC25 3354 GTTCTGAAATGTTCTTAAA  7 TTTAAGAACATTTCAGAAC 26 3735ACTCCAACGTATGTGGTTA  8 TAACCACATACGTTGGAGT 27 3739 CAACGTATGTGGTTATCTG 9 CAGATAACCACATACGTTG 28 3875 TGGGTTAAGTGTTTATCAT 10ATGATAAACACTTAACCCA 29 4153 CATTCTCTATGTACTATGT 11 ACATAGTACATAGAGAATG30 4157 CTCTATGTACTATGTATGT 12 ACATACATAGTACATAGAG 31 5049CAACGTAACGATTTCATGA 13 TCATGAAATCGTTACGTTG 32 5057 CGATTTCATGAACGTTATT14 AATAACGTTCATGAAATCG 33 5058 GATTTCATGAACGTTATTA 15TAATAACGTTCATGAAATC 34 5059 ATTTCATGAACGTTATTAT 16 ATAATAACGTTCATGAAAT35 5108 CTGTATGGGAGCTTAACTT 17 AAGTTAAGCTCCCATACAG 36 5144TGACACTGGTATCTTATTA 18 TAATAAGATACCAGTGTCA 37 5149 CTGGTATCTTATTAAAGTA19 TACTTTAATAAGATACCAG 38

TABLE 3 EPAS1 RNAi agent sequences SEQ SEQ Position ID ID NM_001430Sense NO: Antisense NO:  842 AUGGCGACAUGAUCUUUCU 39 AGAAAGAUCAUGUCGCCAU58 2802 AAAUGUACCCAAUGAUAAG 40 CUUAUCAUUGGGUACAUUU 59 3040GAACUGACCAGAUAUGACU 41 AGUCAUAUCUGGUCAGUUC 60 3304 AGAUGCUCACUUUAUUAUC42 GAUAAUAAAGUGAGCAUCU 61 3310 UCACUUUAUUAUCCCUAUU 43AAUAGGGAUAAUAAAGUGA 62 3345 GUUUUACCUGUUCUGAAAU 44 AUUUCAGAACAGGUAAAAC63 3354 GUUCUGAAAUGUUCUUAAA 45 UUUAAGAACAUUUCAGAAC 64 3735ACUCCAACGUAUGUGGUUA 46 UAACCACAUACGUUGGAGU 65 3739 CAACGUAUGUGGUUAUCUG47 CAGAUAACCACAUACGUUG 66 3875 UGGGUUAAGUGUUUAUCAU 48AUGAUAAACACUUAACCCA 67 4153 CAUUCUCUAUGUACUAUGU 49 ACAUAGUACAUAGAGAAUG68 4157 CUCUAUGUACUAUGUAUGU 50 ACAUACAUAGUACAUAGAG 69 5049CAACGUAACGAUUUCAUGA 51 UCAUGAAAUCGUUACGUUG 70 5057 CGAUUUCAUGAACGUUAUU52 AAUAACGUUCAUGAAAUCG 71 5058 GAUUUCAUGAACGUUAUUA 53UAAUAACGUUCAUGAAAUC 72 5059 AUUUCAUGAACGUUAUUAU 54 AUAAUAACGUUCAUGAAAU73 5108 CUGUAUGGGAGCUUAACUU 55 AAGUUAAGCUCCCAUACAG 74 5144UGACACUGGUAUCUUAUUA 56 UAAUAAGAUACCAGUGUCA 75 5149 CUGGUAUCUUAUUAAAGUA57 UACUUUAAUAAGAUACCAG 76All of these sequences are Homo sapiens sequences.

As noted above, one of the selection criteria for EPAS1 RNAi agents wascross-reactivity between the human and Cyno sequences. If the sequencesare cross-reactive, then the same RNAi agent of the same sequence couldbe used in both Cyno and human experiments. Table 4 indicates thecross-reactivity of various EPAS1 RNAi agents with mouse (Mu), rat (Ra)and Rhesus [mmu or Macaca mulatta endothelial PAS domain protein 1,transcript variant 3 (EPAS1), mRNA].

TABLE 4 Cross-reactivity of EPAS1 RNAi agents. CMatch-REFSEQCMatch-REFSEQ CMatch-REFSEQ NM_010137 NM_023090 XM_001112947Position_NM_001430 Mu(refseq_rna_mm} Ra(refseq_rna_rn) (refseq_rna_mmu)842 Y 2802 3040 Y Y Y 3304 Y 3310 Y 3345 Y 3354 Y 3735 Y 3739 Y 3875 Y4153 4157 Y 5049 Y 5057 5058 5059 5108 Y 5144 Y 5149 Y

All the duplexes tested have Homo sapiens (“hs”) sequences, though forsome of these, the sequence also matches that of the corresponding mouse(Mu or mm, Second column), rat (Ra or rrn, Third column) or Rhesus (mmuor Macaca mulatta, Fourth column) sequence. A “Y” in the appropriatecolumn and row indicates that this sequence matches between human andthe indicated animal. A sequence matching between human and a testanimal allows the same sequence to be used in both animal and humantesting.

Modifications of the duplexes in Table 3 are easily conceived by one ofskill in the art. Example and non-limiting modifications of theseduplexes were conceived. These are listed in Table 3. Additionalmodifications are contemplated.

For the modified variants listed in Table 5, some modifications wereplaced at sites predicted to be sensitive to endonucleases. Somemodifications were designed to eliminate an immune response to the siRNAwhile preserving activity. In general, the sense strand was heavilymodified, and the antisense strand lightly modified. Some modificationsserve more than one purpose. Table 3 lists RNAi agents prepared withthese modifications. In addition, the modification of addition of aterminal dinucleotide dTdT is provided.

TABLE 5 (comprising TABLES 5A to 5E) list different sets of modifiedvariants of the RNAi agent sequences.

Table 5. EPAS1 RNAi Agents (Example Modified Variants).

TABLE 5A EPAS1 RNAi agents (example modified variants). TABLE 5A SEQ IDSEQ ID Position_NM_001430 MODIFIED_SENSE_SEQUENCE NO:MODIFIED_ANTISENSE_SEQUENCE NO: 842 A pU004 pG pG pC pG pA pC pA pU004145 A pG pA pA pA pG pA pU004 126 pG pA pU004 pC pU004 pU004 pU004 pC004pA pU004 pG pU004 pC pG pC pU004 pU004 pu004 pC004 pC004 pA pU004 pU004pU004 2802 A pA pA pU004 pG pU004 pA pC pC pC 146 C pU pU004 pA pU004pC004 pA 127 pA pA pU004 pG pA pU004 pA pA pG pU004 pU004 pG pG pG pU004pA pU004 pU004 pC004 pA pU004 pU004 pU004 pU004 pU004 3040 G pA pA pCpU004 pG pA pC pC pA pG 147 A pG pU004 pC004 pA pU004 pA 128 pA pU004 pApU004 pG pA pC pU004 pU004 pC004 pU004 pG pG pU004 pU004 pU004 pC pA pGpU004 pU004 pC004 pU004 pU004 3304 A pG pA pU004 pG pC pU004 pC pA pC148 G pA pU004 pA pA pU004 pA pA 129 pU004 pU004 pU004 pA pU004 pU004 pApG pU004 pG pA pG pC004 pA pA pU004 pC pU004 pU004 pU004 pC004 pU004pU004 pU004 3310 U004 pC pA pC pU004 pU004 pU004 pA 149 A pA pU004 pA pGpG pG pA 130 pU004 pU004 pA pU004 pC pC pC pU004 pA pA pU004 pA pA pA pGpU004 pA pU004 pU004 pU004 pU004 pU004 pG pA pU004 pU004 3345 G pU004pU004 pU004 pU004 pA pC pC 150 A pU pU004 pU004 pC004 pA pG 131 pU004 pGpU004 pU004 pC pU004 pG pA pA pC004 pA pG pG pU pA pA pA pA pA pU004pU004 pU004 pA pA pC004 pU004 pU004 3354 G pU004 pU004 pC pU004 pG pA pApA 151 U pU pU004 pA pA pG pA pA 132 pU004 pG pU004 pU004 pC pU004 pC004pA pU004 pU004 pU004 pC pU004 pA pA pA pU004 pU004 pA pG pA pA pC004pU004 pU004 3735 A pC pU004 pC pC pA pA pC pG pU004 152 U pA pA pC004pC004 pA pC004 133 pA pU004 pG pU004 pG pG pU004 pA pU004 pA pC004 pGpU004 pU pU004 pA pU004 pU004 pG pG pA pG pU004 pU004 pU004 3739 C pA pApC pG pU004 pA pU004 pG 153 C pA pG pA pU004 pA pA pC004 134 pU004 pG pGpU004 pU004 pA pU004 pC004 pA pC004 pA pU004 pA pC pU004 pG pU004 pU004pC004 pG pU004 pU004 pG pU004 pU004 3875 U004 pG pG pG pU004 pU004 pA pApG 154 A pU pG pA pU004 pA pA pA 135 pU004 pG pU004 pU004 pU004 pA pC004pA pC004 pU004 pU004 pA pU004 pC pA pU004 pU004 pU004 pA pC004 pC004pC004 pA pU004 pU004 4153 C pA pU004 pU004 pC pU004 pC pU004 155 A pC pApU004 pA pG pU004 pA 136 pA pU004 pG pU004 pA pC pU004 pA pC004 pA pU004pA pG pA pG pA pU004 pG pU004 pU004 pU004 pA pU004 pG pU004 pU004 4157 CpU004 pC pU004 pA pU004 pG pU004 156 A pC pA pU004 pA pC004 pA 137 pA pCpU004 pA pU004 pG pU004 pA pU004 pA pG pU004 pA pC004 pA pU004 pG pU004pU004 pU004 pU004 pA pG pA pG pU004 pU004 5049 C pA pA pC pG pU004 pA pApC pG pA 157 U pC pA pU004 pG pA pA pA 138 pU004 pU004 pU004 pC pA pU004pG pU004 pC004 pG pU004 pU004 pA pA pU004 pU004 pC004 pG pU004 pU004 pGpU004 pU004 5057 C pG pA pU004 pU004 pU004 pC pA 158 A pA pU004 pA pApC004 pG 139 pU004 pG pA pA pC pG pU004 pU004 pU004 pU004 pC004 pA pU004pG pA pU004 pU004 pU004 pU004 pA pA pA pU004 pC004 pG pU004 pU004 5058 GpA pU004 pU004 pU004 pC pA pU004 159 U pA pA pU004 pA pA pC004 pG 140 pGpA pA pC pG pU004 pU004 pA pU004 pU004 pC004 pA pU004 pG pU004 pU004 pApU004 pU004 pA pA pA pU004 pC004 pU004 pU004 5059 A pU004 pU004 pU004 pCpA pU004 pG 160 A pU pA pA pU004 pA pA pC004 141 pA pA pC pG pU004 pU004pA pU004 pG pU004 pU004 pC004 pA pU pG pU004 pA pU004 pU004 pU004 pA pApA pU004 pU004 pU004 5108 C pU004 pG pU004 pA pU004 pG pG pG 161 A pA pGpU004 pU004 pA pA pG 142 pA pG pC pU004 pU004 pA pA pC pC004 pU004 pC004pC004 pC004 pU004 pU004 pU004 pU004 pA pU004 pA pC004 pA pG pU004 pU0045144 U004 pG pA pC pA pC pU004 pG pG 162 U pA pA pU004 pA pA pG pA 143pU004 pA pU004 pC pU004 pU004 pA pU004 pA pC004 pC004 pA pG pU004 pU004pA pU004 pU004 pU004 pG pU004 pC004 pA pU004 pU004 5149 C pU004 pG pGpU004 pA pU004 pC 163 U pA pC004 pU004 pU004 pU004 144 pU004 pU004 pApU004 pU004 pA pA pA pA pU004 pA pA pG pA pU pA pA pG pU004 pA pU004pU004 pC004 pC004 pA pG pU004 pU004Table 5A lists example modified variants with the A51 S26 modifications:A51: In guide strand all U as 2′-OMe-U and all C as 2′-OMe-C, exceptpos. 1, 2 and 14; 3′ overhangs as 2′-OMe-U 2′-OMe-US26: In sense strand all U as 2′-OMe-U and all C as 2′-OMe-C; 3′overhangs as 2′-OMe-U 2′-OMe-U“G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, thymidine and uracil as a base,respectively.

FIG. 1 illustrates various example modified nucleotides which have beenor can be used in modified variants of EPAS1 RNAi agents: U002, U003,U004, U005, C004, C005, A004, A005, G005, and G004, which can be used inthe RNAi agents disclosed herein, U002 indicates a 2′-deoxy-thymidinewhich is DNA. U003 indicates 2% deoxy uridine. U004 indicates anucleotide with a Uridine (“U”) base with a 2′-O-methyl modification.U005 indicates a U base with a 2′-O-methoxyethyl (MOE) modification.C004 indicates a Cytosine (“C”) base with a 2′-O-methyl modification.C005 indicates a C base with 2′-O-methoxyethyl modification. A004indicates an Adenosine (“A”) base with a 2′-O-methyl modification. A005indicates an A base with 2′-O-methoxyethyl modification. G005 indicatesa Guanosine (“G”) base with a 2′O-methyl modification. G004 indicates aG base with a 2′O-methyl modification.

“p” indicates phosphate.

TABLE 5B EPAS1 RNAi agents (example modified variants). Duplex SenseConcept Antisense Concept Concept modified-sequence- modified-sequence-Position_NM_001430 nickname_A85S26 string_ A85S26 string_ A85S26 842Homo ApU004 pGpGpC004 pGpApC004 77 ApGpApApApGpApU004 96 Sapiens_(—)pApU004 pGpApU004 pC004 pU004 pCpApU004 pGpU004 EPAS1_(—) pU004 pU004pC004 pU004 pU004 pCpGpCpCpApU004 pU004 842_(—) pU004 pU004 A85_S26 2802Homo ApApApU004 pGpU004 pApC004 78 CpUpU004 pApU004 97 Sapiens_(—) pC004pC004 pApApU004 pCpApU004 pU004 EPAS1_(—) pGpApU004 pApApGpU004 pU004pGpGpGpU004 pApCpApU004 2802_(—) pU004 pU004 pU004 pU004 A85_S26 3040Homo GpApApC004 pU004 pGpApC004 79 ApGpU004 pCpApU004 98 Sapiens_(—)pC004 pApGpApU004 pApU004 pApU004 pCpU004 EPAS1_(—) pGpApC004 pU004pU004 pU004 pGpGpU004 pCpApGpU004 3040_(—) pU004 pCpU004 pU004 A85_S263304 hs_EPAS1_(—) ApGpApU004 pGpC004 pU004 80 GpApU004 pApApU004 993304_(—) pC004 pApC004 pU004 pU004 pApApApGpU004 A85_S26 pU004 pApU004pU004 pApU004 pGpApGpCpApU004 pCpU004 pC004 pU004 pU004 pU004 pU004 3310Homo U004 pC004 pApC004 pU004 81 ApApU004 pApGpGpGpApU004 100Sapiens_(—) pU004 pU004 pApU004 pU004 pApApU004 pApApApGpU004 EPAS1_(—)pApU004 pC004 pC004 pC004 pGpApU004 pU004 3310_(—) pU004 pApU004 pU004pU004 A85_S26 pU004 3345 Homo GpU004 pU004 pU004 pU004 82 ApUpU004 pU004101 Sapiens_(—) pApC004 pC004 pU004 pGpU004 pCpApGpApApCpApGpGpUpApAEPAS1_(—) pU004 pC004 pU004 pApApCpU004 pU004 3345_(—) pGpApApApU004pU004 pU004 A85_S26 3354 Homo GpU004 pU004 pC004 pU004 83 UpUpU004 102Sapiens_(—) pGpApApApU004 pGpU004 pU004 pApApGpApApCpApU004 EPAS1_(—)pC004 pU004 pU004 pApApApU004 pU004 pU004 3354_(—) pU004pGpApGpApApUpU004 pU004 A85_S26 3735 hs_EPAS1_(—) ApC004 pU004 pC004pC004 84 UpApApCpCpApCpApU004 103 3735_(—) pApApC004 pGpU004 pApU004pApCpGpU004 A85_S26 pGpU004 pGpGpU004 pU004 pUpGpGpApGpU004 pU004pApU004 pU004 pU004 3739 Homo C004 pApApC004 pGpU004 85 CpApGpApU004 104Sapiens_(—) pApU004 pGpU004 pGpGpU004 pApApCpCpApGpApU004 EPAS1_(—)pU004 pApU004 pC004 pU004 pApCpGpU004 pU004 3739_(—) pGpU004 pU004pGpU004 pU004 A85_S26 3875 hs_EPAS1_(—) U004 pGpGpGpU004 pU004 86ApUpGpApU004 105 3875_(—) pApApUpU004 pGpU004 pU004 pApApApGpApGpU004pU004 A85_S26 pU004 pApU004 pC004 pApU004 pApApCpCpCpApU004 pU004 pU004pU004 4153 Homo C004 pApU004 pU004 pC004 87 ApCpApU004 pApGpU004 106Sapiens pU004 pC004 pU004 pApU004 pApCpApU004 EPAS1_(—) pGpU004 pApC004pU004 pApU004 pApGpApGpApApU004 4153_(—) pGpU004 pU004 pU004 pGpU004pU004 A85_S26 4157 Homo C004 pU004 pC004 pU004 88 ApCpApU004 pApCpApU004107 Sapiens_(—) pApU004 pGpU004 pApC004 pU004 pApGpU004 pApCpApU004EPAS1_(—) pApU004 pGpU004 pApU004 pApGpApGpU004 pU004 4157_(—) pGpU004pU004 pU004 A85_S26 5049 hs_EPAS1_(—) C004 pApApC004 pGpU004 89UpCpApU004 pGpApApApU004 108 5049_(—) pApApC004 pGpApU004 pU004pCpGpU004 pU004 A85_S26 pU004 pC004 pApU004 pGpApU004 pApCpGpU004 pU004pU004 pGpU004 pU004 5057 Homo C004 pGpApU004 pU004 pU004 90 ApApU004pApApCpGpU004 109 Sapiens_(—) pC004 pApU004 pGpApApC004 pU004 pCpApU004EPAS1_(—) pGpU004 pU004 pApU004 pU004 pGpApApApU004 pCpGpU004 5057_(—)pU004 pU004 pU004 A85_S26 5058 Homo GpApU004 pU004 pU004 pC004 91UpApApU004 pApApCpGpU004 110 Sapiens_(—) pApU004 pGpApApC004 pGpU004pU004 pCpApU004 EPAS1_(—) pU004 pApU004 pU004 pApU004 pGpApApApU004pCpU004 5058_(—) pU004 pU004 A85_S26 5059 Homo ApU004 pU004 pU004 pC00492 ApUpApApC004 111 Sapiens_ EPAS1_(—) pApU004 pGpApApC004 pGpU004pApApCpGpU004 pU004 5059_(—) pU004 pApU004 pU004 pApU004pCpApUpGpApApApU004 A85_S26 pU004 pU004 pU004 pU004 5108 Homo C004 pU004pGpU004 pApU004 93 ApApGpU004 pU004 112 Sapiens_(—) pGpGpGpApGpC004pU004 pU004 pApApGpCpU004 EPAS1_(—) pApApC004 pU004 pU004 pU004pCpCpCpApU004 5108_(—) pU004 pApCpApGpU004 pU004 A85_S26 5144 Homo U004pGpApC004 pApC004 pU004 94 UpApApU004 pApApGpApU004 113 Sapiens_(—)pGpGpU004 pApU004 pC004 pU004 pApCpCpApGpU004 pGpU004 EPAS1_(—) pU004pApU004 pU004 pApU004 pCpApU004 pU004 5144_(—) pU004 A85_S26 5149hs_EPAS1_(—) C004 pU004 pGpGpU004 pApU004 95 UpApCpC004 pU004 pU004 1145149_(—) pC004 pU004 pU004 pApU004 pApApU004 A85_S26 pU004 pApApApGpU004pApU004 pApApGpApUpApCpCpApGpU004 PU004 pU004Table 5B lists example modified variants with the A85 S26 modifications:A85: In guide strand all U as 2′-OMe-U, except positions 1, 2 and 14: 3′overhangs as 2′-OMe-U2′-OMe-US26: In sense strand all U as 2′-OMe-U and all C as 2′-OMe-C: 3′overhangs as 2′-OMe-U 2′-OMe-U

In the duplexes listed in this table, the nickname comprises the termHomo sapiens or “hs”, indicating the source of the sequence. Theposition is also indicated. Also indicated is the modification scheme(e.g., “A85 S26”). This modification scheme is:

A85: In guide strand all U as 2′-OMe-U, except pos. 1, 2 and 14: 3′overhangs as 2′-OMe-U 2′-OMe-U

S26: In sense strand all U as 2′-OMe-U and all C as 2′-OMe-C; 3′overhangs as 2′-OMe-U 2′-OMe-U

In addition, in the nicknames, the underscores and spaces (“_”) areirrelevant. The modifications designated “004” are also diagrammed inFIG. 1.

The duplexes were prepared, as described in Example 2.

Additional example modified variants of EPAS1 sequences are shown inTABLES 5C to 5E, below.

TABLE 5C EPAS1 RNAi agents (example modified variants) Duplex SEQ ID SEQID Pos. nickname MODIFIED_ SENSE_ SEQUENCE NO: MODIFIED_ ANTISENSE_SEQUENCE NO: 842 hs_(—) A pU004 pG pG pC pG pA pC pA 164 A pG pA pA pApG pA pU004 182 EPAS1_(—) pU004 pG pA pU004 pC pU004 pC004 pA pU004 pGpU004 pC 842_(—) pU004 pU004 pC pU004 pU004 pG pC004 pC004 pA pU004A51S53 pU004 pU004 pU004 2802 hs_(—) A pA pA pU004 pG pU004 pA pC pC 165C pU pU004 pA pU004 pC004 183 EPAS1_(—) pC pA pA pU004 pG pA pU004 pA pApU004 pU004 pG pG pG 2802_(—) pA pG pU004 pU004 pU004 pA pC004 pA pU004A51S53 pU004 pU004 pU004 pU004 3040 hs_(—) G pA pA pC pU004 pG pA pC pCpA 166 A pG pU004 pC004 pA pU004 184 EPAS1_(—) pG pA pU004 pA pU004 pGpA pC pA pU004 pC004 pU004 pG pG 3040_(—) pU004 pU004 pU004 pU004 pC pApG pU004 pU004 A51S53 pC004 pU004 pU004 3304 hs_(—) A pG pA pU004 pG pCpU004 pC pA 167 G pA pU004 pA pA pU004 pA 185 EPAS1_(—) pC pU004 pU004pU004 pA pU004 pA pA pG pU004 pG pA pG 3304_(—) pU004 pA pU004 pC pU004pU004 pC004 pA pU004 pC004 pU004 A51S53 pU004 pU004 3310 hs_(—) U004 pCpA pC pU004 pU004 pU004 168 A pA pU004 pA pG pG pG pA 186 EPAS1_(—) pApU004 pU004 pA pU004 pC pC pU004 pA pA pU004 pA pA pA 3310_(—) pC pU004pA pU004 pU004 pU004 pG pU004 pG pA pU004 pU004 A51S53 pU004 3345 hs_(—)G pU004 pU004 pU004 pU004 pA pC 169 A pU pU004 pU004 pC004 pA 187EPAS1_(—) pC pU004 pG pU004 pU004 pC pG pA pA pC004 pA pG pG pU 3345_(—)pU004 pG pA pA pA pU004 pU004 pA pA pA pA pC004 pU004 A51S53 pU004 pU0043354 hs_(—) G pU004 pU004 pC pU004 pG pA pA 170 U pU pU004 pA pA pG pApA 188 EPAS1_(—) pA pU004 pG pU004 pU004 pC pC004 pA pU004 pU004 pU0043354_(—) pU004 pU004 pA pA pA pU004 pC pA pC pA pA pC004 pU004 A51S53pU004 pU004 3735 hs_(—) A pC pU004 pC pC pA pA pC pG 171 U pA pA pC004pC004 pA 189 EPAS1_(—) pU004 pA pU004 pG pU004 pG pG pC004 pA pU004 pApC004 pG 3735_(—) pU004 pU004 pA pU004 pU004 pU004 pU pG pG pA pG pU004A51S33 pU004 pU004 3739 hs_(—) C pA pA pC pG pU004 pA pU004 pG 172 C pApG pA pU004 pA pA 190 EPAS1_(—) pU004 pG pG pU004 pU004 pA pC004 pC004pA pC004 pA 3739_(—) pU004 pC pU004 pG pU004 pU004 pU004 pA pC004 pGpU004 A51S53 pU004 pG pU004 pU004 3875 hs_(—) U004 pG pG pG pU004 pU004pA pA 173 A pU pG pA pU004 pA pA pA 191 EPAS1_(—) pG pU004 pG pU004pU004 pU004 pC004 pA pC004 pU004 pU004 3875_(—) pA pU004 pC pA pU004pU004 pA pA pC004 pC004 pC004 pA A51S53 pU004 pU004 pU004 4157 hs_(—) CpU004 pC pU004 pA pU004 pG 174 A pC pA pU004 pA pC004 pA 192 EPAS1_(—)pU004 pA pC pU004 pA pU004 pG pU004 pA pG pU004 pA pC004 4157_(—) pU004pA pU004 pG pU004 pU004 pA pU004 pA pG pA pG pU004 A51S53 pU004 pU0045049 hs_(—) C pA pA pC pG pU004 pA pA pC pG 175 U pC pA pU004 pG pA pApA 193 EPAS1_(—) pA pU004 pU004 pU004 pC pA pU004 pC004 pG pU004 pU0045049_(—) pU004 pG pA pU004 pU004 pA pC004 pG pU004 pU004 pG A51S53 pU004pU004 5057 hs_(—) C pG pA pU004 pU004 pU004 pC pA 176 A pA pU004 pA pApC004 pG 194 EPAS1_(—) pU004 pG pA pA pC pG pU004 pU004 pU004 pC004 pApU004 5057_(—) pU004 pA pU004 pU004 pU004 pG pA pA pA pU004 pC004 pGA51S53 pU004 pU004 pU004 5058 hs_(—) G pA pU004 pU004 pU004 pC pA 177 UpA pA pU004 pA pA pC004 195 EPAS1_(—) pU004 pG pA pA pC pG pU004 pGpU004 pU004 pC004 pA 5058_(—) pU004 pA pU004 pU004 pA pU004 pU004 pG pApA pA pU004 A51S53 pU004 pC004 pU004 pU004 5059 hs_(—) A pU004 pU004pU004 pC pA pU004 178 A pU pA pA pU004 pA pA 196 EPAS1_(—) pG pA pA pCpG pU004 pU004 pA pC004 pG pU004 pU004 pC004 5059_(—) pU004 pU004 pApU004 pU004 pA pU pG pA pA pA pU004 A51S53 pU004 pU004 pU004 5108 hs_(—)C pU004 pG pU004 pA pU004 pG pG 179 A pA pG pU004 pU004 pA pA 197EPAS1_(—) pG pA pG pC pU004 pU004 pA pA pG pC004 pU004 pC004 pC0045108_(—) pC pU004 pU004 pU004 pU004 pC004 pA pU004 pA pC004 pA A51S53 pGpU004 pU004 5144 hs_(—) U004 pG pA pC pA pC pU004 pG pG 180 U pA pApU004 pA pA pG pA 198 EPAS1_(—) pU004 pA pU004 pC pU004 pU004 pU004 pApC004 pC004 pA pG 5144_(—) pA pU004 pU004 pA pU004 pU004 pU004 pG pU004pC004 pA A51S53 pU004 pU004 5149 hs_(—) C pU004 pG pG pU004 pA pU004 pC181 U pA pC004 pU004 pU004 199 EPAS1_(—) pU004 pU004 pA pU004 pU004 pApU004 pA pA pU004 pA pA pG 5149_(—) pA pA pG pU004 pA pU004 pU004 pA pUpA pC004 pC004 pA pG A51S53 pU004 pU004Table 5C presents additional modified variants with the A51S53modifications. Position (“Pos.”) is provided, as are the Duplex name andthe sequence of the example modified sense and antisense sequence. 004(2′-OMe) and other terms are defined elsewhere herein and in FIG. 1.

TABLE 5D EPAS1 RNAi agents (example modified variants) SEQ SEQ ID IDNickname_siRNA 19-mer_guide_modified NO: 19-mer_sense_mod NO:hs_EPAS1_842_A51S53 AGAAAGAtcAtGtCGccAt 200 AtGGCGACAtGAtCtttCt 225hs_EPAS1_2802_A51S53 CTtAtcAttGGGtAcAttt 201 AAAtGtACCCAAtGAtAAG 226hs_EPAS1_3040_A51S53 AGtcAtAtctGGtCAGttc 202 GAACtGACCAGAtAtGACt 227hs_EPAS1_3304_A51S53 GAtAAtAAAGtGAGcAtct 203 AGAtGCtCAVtttAttAtC 228hs_EPAS1_3310_A51S53 AAtAGGGAtAAtAAAGtGA 204 tCACtttAttAtCCCtAtt 229hs_EPAS1_3345_A51S53 ATttcAGAAcAGGTAAAAc 205 GttttAACtGttCtGAAAt 230hs_EPAS1_3354_A51S53 TTtAAGAAcAtttCAGAAc 206 GttCtGAAAtGttCttAAA 231hs_EPAS1_3735_A51S53 TAAccAcAtAcGtTGGAGt 207 ACtCCAACGtAtGtGGttA 232hs_EPAS1_3739_A51S53 CAGAtAAccAcAtAcGttG 208 CAACGtAtGtGGttAtCtG 233hs_EPAS1_3742_A51S53 TCAcAGAtAAccACAtAcG 209 CGtAtGtGGttAtCtGtGA 234hs_EPAS1_3743_A51S53 TTcAcAGAtAAccAcAtAc 210 GtAtGtGGttAtCtGtGAA 235hs_EPAS1_3747_A51S53 AActttcAcAGAtAAccAc 211 GtGGttAtCtGtGAAAGtt 236hs_EPAS1_3778_A51S53 AAAcAccAGtttAGGAAAA 212 ttttCCtAAACtGGtGttt 237hs_EPAS1_3870_A51S53 AAAcActtAAcccAGAtAt 213 AtAtCtGGGttAAGtGttt 238hs_EPAS1_3871_A51S53 TAAAcActtAAccCAGAtA 214 tAtCtGGGttAAGtGtttA 239hs_EPAS1_3875_A51S53 ATGAtAAAcActtAAcccA 215 tGGGttAAGtGtttAtCAt 240hs_EPAS1_4153_A51S53 ACAtAGtAcAtAGAGAAtG 216 CAttCtCtAtGtACtAtGt 241hs_EPAS1_4157_A51S53 ACAtAcAtAGtAcAtAGAG 217 CtCtAtGtACtAtGtAtGt 242hs_EPAS1_5049_A51S53 TCAtGAAAtcGttAcGttG 218 CAACGtAACGAtttCAtGA 243hs_EPAS1_5057_A51S53 AAtAAcGttcAtGAAAtcG 219 CGAtttCAtGAACGttAtt 244hs_EPAS1_5058_A51S53 TAAtAAcGttcAtGAAAtc 220 GAtttCAtGAACGttAttA 245hs_EPAS1_5059_A51S53 ATAAtAAcGttcATGAAAt 221 AtttCAtGAACGttAttAt 246hs_EPAS1_5108_A51S53 AAGttAAgctcccAtAcAG 222 CtGtAtGGGAGCttAACtt 247hs_EPAS1_5144_A51S53 TAAtAAGAtAccAGtGtcA 223 tGACACtGGtAtCttAttA 248hs_EPAS1_5149_A51S53 TActttAAtAAGATAccAG 224 CtGGtAtCttAttAAAGtA 249Table 5D presents example modified EPAS1 RNAi agents, shown as 19-mermodified sequences. Table 5E shows related sequences as 21-mer sequences(including a dinucleotide overhang).

TABLE 5E EPAS1 RNAi agents (example modified variants) SEQ SEQ ID IDNickname_siRNA 21-mer_guide_modified NO: 21-mer_sense_mod NO:hs_EPAS1_842_A51S53 AGAAAGAtcAtGtCGccAtuu 250 AtGGCGACAtGAtCtttCtuu 275hs_EPAS1_2802_A51S53 CTtAtcAttGGGtAcAtttuu 251 AAAtGtACCCAAtGAtAAGuu 276hs_EPAS1_3040_A51S53 AGtcAtAtctGGtCAGttcuu 252 GAACtGACCAGAtAtGACtuu 277hs_EPAS1_3304_A51S53 GAtAAtAAAGtGAGcAtctuu 253 AGAtGCtCACtttAttAtCuu 278hs_EPAS1_3310_A51S53 AAtAGGGAtAAtAAAGtGAuu 254 tCACtttAttAtCCCtAttuu 279hs_EPAS1_3345_A51S53 ATttcAGAAcAGGTAAAAcuu 255 GttttACCtGttCtGAAAtuu 280hs_EPAS1_3354_A51S53 TTtAAGAAcAtttCAGAAcuu 256 GttCtGAAAtGttCttAAAuu 281hs_EPAS1_3735_A51S53 TAAccAcAtAcGtTGGAGtuu 257 ACtCCAACGtAtGtGGttAuu 282hs_EPAS1_3739_A51S53 CAGAtAAccAcAtAcGttGuu 258 CAACGtAtGtGGttAtCtGuu 283hs_EPAS1_3742_A51S53 TCAcAGAtAAccACAtAcGuu 259 CGtAtGtGGttAtCtGtGAuu 284hs_EPAS1_3743_A51S53 TTcAcAGAtAAccAcAtAcuu 260 GtAtGtGGttAtCtGtGAAuu 285hs_EPAS1_3747_A51S53 AActttcAcAGAtAAccAcuu 261 GtGGttAtCtGtGAAAGttuu 286hs_EPAS1_3778_A51S53 AAAcAccAGtttAGGAAAAuu 262 ttttCCtAAACtGGtGtttuu 287hs_EPAS1_3870_A51S53 AAAcActtAAcccAGAtAtuu 263 AtAtCtGGGttAAGtGtttuu 288hs_EPAS1_3871_A51S53 TAAAcActtAAccCAGAtAuu 264 tAtCtGGGttAAGtGtttAuu 289hs_EPAS1_3875_A51S53 ATGAtAAAcActtAAcccAuu 265 tGGGttAAGtGtttAtCAtuu 290hs_EPAS1_4153_A51S53 ACAtAGtAcAtAGAGAAtGuu 266 CAttCtCtAtGtACtAtGtuu 291hs_EPAS1_4157_A51S53 ACAtAcAtAGtAcAtAGAGuu 267 CtCtAtGtACtAtGtAtGtuu 292hs_EPAS1_5049_A51S53 TCAtGAAAtcGttAcGttGuu 268 CAACGtAACGAtttCAtGAuu 293hs_EPAS1_5057_A51S53 AAtAAcGttcAtGAAAtcGuu 269 CGAtttCAtGAACGttAttuu 294hs_EPAS1_5058_A51S53 TAAtAAcGttcAtGAAAtcuu 270 GAtttCAtGAACGttAttAuu 295hs_EPAS1_5059_A51S53 ATAAtAAcGttcATGAAAtuu 271 AtttCAtGAACGttAttAtuu 296hs_EPAS1_5108_A51S53 AAGttAAGctcccAtAcAGuu 272 CtGtAtGGGAGCttAACttuu 297hs_EPAS1_5144_A51S53 TAAtAAGAtAccAGtGtcAuu 273 tGACACtGGtAtCttAttAuu 298hs_EPAS1_5149_A51S53 TActttAAtAAGATAccAGuu 274 CtGGtAtCttAttAAAGtAuu 299Table 5E presents example modified EPAS1 RNAi agents, shown as 21-mermodified sequence (including a dinucleotide overhang). Table 5D showsrelated sequence as 21-mer sequencesAdditional Modified Sequences

Below are listed modified variants of EPAS1 RNAi agent sequences:

siRNA 5049 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Guide U C A uG A A A u c G u u A c G u u G u u strand Sense u u A G u A C u u u A G CA A u G C A A C strandA modified variant of EPAS1 RNAi agent 5049 [wherein hold fontnucleotides at positions 4, 9, 10, 12, 13, 15, 17 and 18 (counting 5′ to3′) in the Guide Strand, and 6, 12-14, 17, and 20-21 (counting 5′ to 3′)in the Sense strand are 2′-OMe] is presented above, wherein the Guide(Anti-sense) strand is SEQ ID NO: 300 and the sense strand is SEQ ID NO:301.

siRNA 3875 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Guide A U G Au A A A C A C u u A A C C C A u u strand Sense u u u A c u A u u u G u GA A u u G G G u strandA modified variant of EPAS1. RNAi agent 3875 [wherein bold fontnucleotides at positions 5, 12, 13, and 20-21 (counting 5′ to 3′) in theGuide strand, and positions 1, 5-6, 10, 12-14, 16-17, and 19-21(counting 5′ to 3′) in the Sense strand are 2′-OMe] is presented above,wherein the Guide (Anti-sense) strand is SEQ ID NO: 302 and the sensestrand is SEQ ID NO: 305.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Guide U C A u G A A A uc G u u A c G u u G A C strand Sense C A A G u A C u u u A G C A A u G CA A C strandA modified variant of EPAS1 RNAi agent 5049 [wherein hold frontnucleotides at positions 4, 9-10, 12-13, 15, and 17-18 (counting 5′ to3′) in the Guide Strand, and positions 6, 12-14, and 17 (counting 5′ to3′) in the Sense Strand are 2′-OMe] is presented above, wherein theGuide (Anti-sense) strand is SEQ ID NO: 303 and the sense strand is SEQID NO: 304.Lower case nt (e.g., “u” and “c”) are 2′-OMe. It should be understoodthat the sequences as represented herein (e.g., SEQ ID NO: 303 or nt1-19 or SEQ ID NO: 303) represent both modified and unmodified variants.Additional modified variants of the EPAS1 RNAi agent sequences disclosedherein can be readily produced by one of ordinary skill in the art.

Example 2. Preparation of siRNAs

Small scale synthesis is used to prepare EPAS1 siRNAs; medium and largescale syntheses can also be used to prepare these siRNAs in largerquantities.

Small Scale Synthesis and Purification Methods for the Initial Screens(1 μMole Scale).

Small scale synthesis is used to generate siRNAs.

EPAS1 sequences are synthesized on MerMade 192 synthesizer (BioAutomation, Plano, Tex.) at 1 μmol scale.

In some experiments, for all the sequences in the list, ‘endolight’chemistry is applied as detailed below: All pyrimidines (cytosine anduridine) in the sense strand contain 2′-O-Methyl bases (2′ O-Methyl Cand 2′-O-Methyl U). In the antisense strand, pyrimidines adjacent to(towards 5′ position) ribo A nucleoside are replaced with theircorresponding 2-O-Methyl nucleotides.

In some experiments, a two base dTdT extension at 3′ end of both senseand antisense sequences is introduced.

The sequence file is converted to a text file to make it compatible forloading in the MerMade 192 synthesis software.

Synthesis, Cleavage and Deprotection:

The synthesis of EPAS1 sequences can use solid supported oligonucleotidesynthesis phosphoramidite chemistry.

The synthesis of the above sequences performed at 1 μM scale in 96 wellplates. The ribo and 2-O-Methyl phosphoramidite solutions are preparedat 0.1M concentration and ethyl thio tetrazole (0.6M in Acetonitrile) isused as activator. Deblock solution, oxidizer solution and cappingsolution are prepared according to standard processes.

The synthesized sequences are cleaved and deprotected in 96 well plates,using methylamine solution (a 3:1 mixture of aqueous and ethanolicsolutions) in the first step and fluoride reagent in the second step.The crude sequences are precipitated using acetone:ethanol (80:20) mixand the pellet are re-suspended in 0.02M sodium acetate buffer. Samplesfrom each sequence are analyzed by LC-MS to confirm the identity, UV forquantification and a selected set of samples by IEX chromatography todetermine purity.

Purification and Desalting:

EPAS1 tiled sequences are purified on AKTA explorer purification systemusing Source 15Q column. A column temperature of 65 C is maintainedduring purification. Sample injection and collection are performed in 96well (1.8 mL-deep well) plates. A single peak corresponding to the fulllength sequence is collected in the eluent. The purified sequences aredesalted on a Sephadex G25 column using AKTA purifier. The concentrationof desalted EPAS1 sequences are calculated using absorbance at 260 nmwavelength and purity was measured by ion exchange chromatography.

Annealing:

Purified desalted sense and antisense single strands are mixed inequimolar amounts and annealed to form EPAS1 duplexes. The duplexes areprepared at 10 uM concentration in 1× PBS buffer and tested by capillarygel electrophoresis for purity.

Medium Scale Synthesis and Purification (1-50 μmol)

Medium scale synthesis can also be used to generate siRNAs.

Single-stranded RNAs in scales between 1 and 50 μmol are prepared bysolid phase synthesis using an ABI DNA/RNA Synthesizer 394 (AppliedBiosystems) and controlled pore glass (CPG, 500 Å, loading 80-100μmol/g) purchased from Prime Synthesis (Aston, Pa.) as the solidsupport. For larger scales, empty synthesis columns (10 μmol) from GlenResearch Corp. and large amidite (80 mL) and reagent bottles (450 mL)are used. RNA and RNA containing 2′-O-methyl nucleotides are generatedby solid phase synthesis employing the corresponding phosphoramiditesand 2′-O-methyl phosphoramidites, respectively (ChemGenes, Wilmington,Mass.). These building blocks are incorporated at selected sites withinthe sequence of the oligoribonucleotide chain using standard nucleosidephosphoramidite chemistry such as described in Current Protocols inNucleic Acid Chemistry. Beaucage, S. L. et al. (Edrs.), John Wiley &Sons. Inc. New York, N.Y., USA. Phosphorothioate linkages are introducedusing a solution of the 0.1 M DDTT (AM Chemicals, Oceanside, Calif.) inpyridine. Further ancillary reagents are obtained from Glen ResearchCorp. (Sterling, Va.).

Deprotection and purification of the crude oligoribonucleotides by anionexchange HPLC are carried out according to established procedures.Yields and concentrations are determined spectrophotometrically at awavelength of 260 nm. Double stranded RNA is generated by mixing anequimolar solution of complementary strands in annealing buffer(typically phosphate buffered solution, PBS, Ambion, Applied Biosystems,Austin, Tex.) at the desired concentration. The mixture is then heatedin a water bath at 85-90° C. for 5 minutes and cooled to roomtemperature over a period of 3-4 hours. The RNA duplex is stored at −20°C. until use.

Example 3 Example 3A. Methodology for In Vitro Screening

Cell Culture and Transfections

In some experiments, 786-O cells are grown to near confluence at 37° C.in an atmosphere of 5% CO₂ in DMEM supplemented with 10% FBS,streptomycin, and glutamine before being released from the flask(s) bytrypsinization. Reverse transfection is carried out by adding 15 μl ofOpti-MEM/siRNA duplexes to 77.5 ul of Opti-MEM plus 2.5 μl ofLipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat#13778-150) into a 384-well plate and incubating at room temperature for20 minutes. 15 ul of this complex is transferred to another 384 wellplate. 2.0×10³ 786-O cells are then added. Cells are incubated for 48hours prior to the addition of Bright-Glo to each well. Single point,experiments use 6.5 nM final duplex concentration for 786-O cells foreach of the EPAS1 siRNAs. A subset of siRNAs that showed robustsilencing in the 6.5 nM screens are assayed over a range ofconcentrations from 10 nM to 0.0006 nM to determine their IC50.

Hundreds of EPAS1 duplexes were tested (Table 6 and data not shown).These demonstrated a wide range of RNAi activity, from poor toexcellent. A subset of 19 EPAS1 RNAi agents is shown in Table 6, below.Additional studies wore performed using other modified variants of theEPAS1 RNAi agents listed herein (data not shown).

TABLE 6. KD (Gene knock-down) media led by EPAS1 RNAI agents.

The EPAS1 RNAi agents used in Table 6 are the modified variants shown inTable 5C. The residual gene activity at 30 nM, 15 nM and 7.5 nM in 786-Ocells and SD (standard deviation) and residual activity at 6 nM in HeLacells and SD are presented. The numbers indicate the residual geneactivity; thus, for 842, column 2 indicates that at 30 nM in 786-Ocells, there was 16.8% residual EPAS1 gene activity (compared tocontrol), or 83.2% gene knockdown (reduction in gene activity).

TABLE 6 KD (Gene knock-down) mediated by EPAS1 RNAI agents. % % %Residual Residual Residual Residual Activity SD Activity SD Activity SDActivity SD 786-O 786-O 786-O 786-O 786-O 786-O HeLa HeLa 30 nM 30 nM 15nM 15 nM 7.5 nM 7.5 nM 6 nM 6 nM Nick- BC: BC: BC: BC: BC: BC: BC: BC:name_siRNA 2739- 2739- 2705- 2705- 2771- 2771- 2713- 2713- OLD 2746 27462712 2712 2778 2778 2720 2720 hs_EPAS1_842_A51S53 16.8 3.2 9.2 0.8 6.63.1 29.1 5.3 hs_EPAS1_2802_A51S53 9.5 0.6 19.2 3.6 12.2 2.6 45.5 13.8hs_EPAS1_3040_A51S53 20.5 8.6 30.4 4 14.4 2.7 56 17.3hs_EPAS1_3304_A51S53 18.4 4 37.8 6 16.9 6.1 47.7 23.6hs_EPAS1_3310_A51S53 19 4 29.2 4.3 12.9 4.5 58.3 22.7hs_EPAS1_3345_A51S53 20.1 7.1 30.4 8.1 9.8 0.9 48.8 13.9hs_EPAS1_3354_A51S53 22.6 9.5 38.3 12.8 10.3 3 50.3 9.3hs_EPAS1_3735_A51S53 22.3 5.4 20.5 3 15.5 7.4 41.4 7.4hs_EPAS1_3739_A51S53 11.9 3.5 24.5 7 17.4 9.6 34.7 14.9hs_EPAS1_3875_A51S53 26.3 6.4 28 2.4 12.5 4.9 47.8 24.9hs_EPAS1_4157_A51S53 13.5 4.1 25.6 3.8 18.8 7.9 29.5 7.2hs_EPAS1_5049_A51S53 17.6 9.7 14.5 5.2 8.3 1.1 18.7 10.7hs_EPAS1_5057_A51S53 11.2 3.5 10.5 2.9 5.2 0.3 26.2 10hs_EPAS1_5058_A51S53 7.7 2.1 12.2 6.7 10.2 1.7 28 14.2hs_EPAS1_5059_A51S53 12.4 3.8 8.9 4.1 6.2 3.1 32.6 15.4hs_EPAS1_5108_A51S53 10.4 4.5 12.8 3.2 8.4 0.6 24.7 16.4hs_EPAS1_5144_A51S53 22.8 2.1 21.6 11 10 2.4 21.9 5.9hs_EPAS1_5149_A51S53 10.5 2.5 22.7 5.7 11.4 4.6 29 10.5

Example 4. EC50 (IC50) Data of EPAS1 RNAi Agents in 786-O Cells(Reporter Assay)

Of the hundreds of EPAS1 duplexes designed, constructed and screened(Example 3 and data not shown), a subset of 19 efficacious EPAS1 RNAiagents were chosen for further study, including determination of EC50.

The purpose of this screen was to determine the EC50 (effectiveconcentration 50; or the minimum dosage of RNAi agent capable ofreducing the luciferase signal by 50%. The term IC50 (inhibitoryconcentration) is used interchangeably with EC50 herein.

Several criteria for selection were used, included, for example, >80% KD(gene knockdown), although in some cases, a particular EPAS1 RNAi agentdid not meet all criteria evaluated. 786-O HRE cells were constructedusing HRE-LUC reporter gene. In these cells, the Luciferase (LUC)reporter gene is under control of the Hif Response Element (HRE).

In addition, RNAi agents are tested for an ability to decrease HRE-lucPEST but do not effect UB6-luc Pest. Successful siRNA candidates had agreater than 100 fold IC-50 window between the on-target HRE-luc PESTassay and the off-target UB6-luc PEST assay. This indicates that thesesiRNA sequences do not cause off-target effects on cell growth,transcription, or translation in the 786-O cell line.

To determine IC50, a serial dilution was used, comprising 8concentrations between 10 nM and 0.0006 nM. The siRNA duplexes weretransfected into cells using Lipofectamine RNAiMax (Zhao et al. 2008Mol. Biotech, 40: 19-26).

TABLE 7A IC50 DATA FOR EPAS1 RNAi AGENTS Position_NM_001430 A85_S26_IC50nM_HRE 842 0.093 2802 0.38 3040 1.07 3304 0.43 3310 0.13 3345 0.12 33450.087 3735 0.102 3739 0.11 3875 0.042 4153 0.083 4157 0.104 5049 0.0755057 0.107 5058 0.14 5059 0.186 5108 0.27 5144 0.085 5149 0.2

Table 7A shows the EC50 (or IC50) of various EPAS1 duplexes. Forexample, in the first line of this table, 842 with the modification setA85 S26 demonstrated in one assay an IC50 of 0.093 nM, after 3 days.

TABLE 7B IC50 DATA FOR EPAS1 RNAi AGENTS LNP A LNP B A51S53 A51S53A51S53 Nick Name IC50 nM IC50 NM IC50 NM HRE 842 A51 S53 0.9 1 0.1 2802A51 S53 1.1 1.9.1 0.37 3040 A51 S53 1 0.89 0.42 3304 A51 S53 1.2 1 3310A51 S53 3345 A51 S53 0.63 0.5 0.19 3354 A51 S53 0.017 3735 A51 S53 0.0793739 A51 S53 0.39 0.29 0.19 3875 A51 S53 4153 A51 S53 0.046 4157 A51 S530.06 5049 A51 S53 0.074 5057 A51 S53 0.35 0.25 0.2 5058 A51 S53 0.830.74 0.2 5059 A51 S53 0.76 1.09 0.13 5108 A51 S53 0.34 0.33 0.21 5144A51 S53 0.05 5149 A51 S53 0.28 0.32 0.18Table 7B shows IC50 data using the EPAS1 RNAi agents with the A51 S53modification set. In columns 2 and 3, the RNAi agents were deliveredwith either of two LNP formulations (lipid nanoparticle formulationsdesignated A and B), and in column 4, naked RNAi agents were used.Successful siRNA candidates had a greater than 100 fold IC-50 windowbetween the on-target HRE-luc PEST assay and the off-target UB6-luc PESTassay. This indicates that these siRNA sequences do not cause off-targeteffects on cell growth, transcription, or translation in the 786-O cellline.

Example 5. In Vivo Data of EPAS1 RNAi Agents

Effect of EPAS1 RNAi Agents on 786-O Tumor Models in Nude Mice.

The efficacy of various EPAS1 RNAi agents in lipid nanoparticles wastested in vivo against 786-O clear cell renal cell carcinoma (CCRCC)tumors in nude mice. The numbers in the Table 8, below, show knockdown;e.g., “0.38” indicates 38% of control.

TABLE 8 Efficacy of EPAS1 RNAi agents against 786-O tumors in nude mice.KD TUMOR TUMOR TUMOR DATA 537 DATA 338 DATA 338 Study 72 Studies 76 & 80Study 88 Nick Name A51 S53 A51 S53 A51 S53 842 A51 S53 0.38 0.21 2802A51 S53 0.46 0.4 3040 A51 S53 0.16 3304 A51 S53 0.43 0.55 3310 A51 S530.35 3345 A51 S53 0.2 0.33 3354 A51 S53 0.3 3735 A51 S53 0.5 0.3 3739A51 S53 0.32 3875 A51 S53 0.4 0.37 4153 A51 S53 0.3 4157 A51 S53 0.35049 A51 S53 0.55 5057 A51 S53 0.4 5058 A51 S53 0.45 0.4 5058 A51 S530.47 0.5 5108 A51 S53 0.3 5144 A51 S53 0.3 5149 A51 S53 0.4Some of these experiments show a decrease in tumor volume, especially inthe late stage (towards the end of the experiment).

In a separate in vivo testing experiment, EPAS1 RNAi agents 5039, 3875and 3735 are tested against 786-O tumors in nude mice.

5049 displays 0.17 (17% residual gene activity, or 83% gene knockdown).

3875 displays 0.24 (24% residual gene activity, or gene knockdown).

3735 displays 0.24 (24 residual gene activity, or 76% gene knockdown).

Example 6. Overlapping RNAi Agents

Some of the siRNAs listed above overlap each other in sequence. Thefollowing table presents a compilation of groups of RNAi agents, whereineach member of a group overlaps with each other member of the same groupley at least 12 nt. A 12-nt portion of the overlap of the sense strandsand a 12-nt portion of the overlap of the antisense strand are presentedalthough in some cases the overlapping portion is longer than 12 nt.Thus, for example, 3304 and 3310 share the common technical feature ofthe sequence of UCACUUUAUUAUC (SEQ ID NO: 115) in the sense strand, andthe sequence of GAUAAUAAAGUGA (SEQ ID NO: 120) in the antisense strand.Note, of course, that various groupings comprise different numbers ofoverlapping siRNAs; any two siRNAs within that group overlap. Thus,5057, 5058 and 5059 all overlap with each other, meaning that any two ofthat group (5057 and 5059; or 5058 and 5059; or 5058 and 5059) share acommon technical feature of an overlapping portion of the sense and/oranti-sense strand sequence. The disclosure thus encompasses any pair orgrouping of overlapping siRNAs, wherein the pair share a technicalfeature, namely, the portion of the sense and/or anti-sense strand whichoverlaps, as described in Table 9.

TABLE 9 OVERLAPPING siRNAs SEQ SEQ Position ID ID NM_001430 Sense NO:Antisense NO: 3304 and  UCACUUUAUUAUC 115 GAUAAUAAAGUGA 120 33103735 and  CAACGUAUGUGGUUA 116 UAACCACAUACGUUG 121 3739 4153 and CUCUAUGUACUAUGU 117 ACAUAGUACAUAGAG 122 4157 5057, 5058AUUUCAUGAACGUUAUU 118 AAUAACGUUCAUGAAAU 123 and 5059 5144 and CUGGUAUCUUAUUA 119 UAAUAAGAUACCAG 124 5149Thus, in various aspects, the disclosure pertains to a group ofoverlapping RNAi agents which comprise or consist or; or which comprisea sense strand and/or anti-sense strand of; or which comprise a sensestrand and/or and anti-sense strand comprising at least 15 contiguous ntwith 0, 1, 2 or 3 mismatches from the sense and/or anti-sense strand of:any grouping of 3304 and 3310; 3735 and 3739; 4153 and 4157; 5057 and5059; or 5058 and 5059; or 5058 and 5059 or 5144 and 5149; andmodifications and variants thereof; and any overlapping pair or groupthereof.

Embodiments

-   1. A composition comprising an RNAi agent comprising a sense strand    and an antisense strand, wherein the antisense strand comprises at    least 15 contiguous nucleotides differing by 0, 1, 2, or 3    nucleotides from the antisense strand of an RNAi agent to EPAS1    provided in any of Tables 1 to 5.-   2. The composition of embodiment 1, wherein the composition further    comprises a second RNAi agent to EPAS1.-   3. The composition of embodiment 1, wherein the antisense strand is    about 30 or fewer nucleotides in length.-   4. The composition of embodiment 1, wherein the sense strand and the    antisense strand form a duplex region about 15 to about 30    nucleotide pairs in length.-   5. The composition of embodiment 1, wherein the antisense strand and    the sense strand are both about 19 to about 23 nucleotides in    length.-   6. The composition of embodiment 1, wherein the RNAi agent comprises    a modification that causes the RNAi agent to have increased    stability in a biological sample or environment.-   7. The composition of embodiment 1, wherein the RNAi agent comprises    a modified sugar backbone, including a phosphorothioate linkage, or    a 2′-modified nucleotide.-   8. The composition of embodiment 1, wherein the RNAi agent    comprises;-   at least one 5′-uridine-adenine-3′ (5′-ua-3′) dinucleotide, wherein    the uridine is a 2′-modified nucleotide; at least one    5′-uridine-guanine-3′ (5′-ug-3′) dinucleotide, wherein the    5′-uridine is a 2′-modified nucleotide; at least one    5′-cytidine-adenine-3′ (5′-ca-3′) dinucleotide, wherein the    5′-cytidine is a 2′-modified nucleotide; or at least one    5′-uridine-uridine-3′ (5′-uu-3′) dinucleotide, wherein the    5′-uridine is a 2′-modified nucleotide.-   9. The composition of embodiment 1, wherein the RNAi agent comprises    a 2′-modification selected from the group consisting of: 2′-deoxy,    2′-deoxy-2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE),    2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE),    2′-O-dimethylaminopropyl (2′-O-DMAP).    2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), and    2′-O—N-methylacetamido (2′-O-NMA).-   10. The composition of embodiment 1, wherein the RNAi agent    comprises a blunt end.-   11. The composition of embodiment 1, wherein the RNAi agent    comprises an overhang having 1 to 4 unpaired nucleotides.-   12. The composition of embodiment 1, wherein the RNAi agent    comprises an overhang at the 3′-end of the antisense strand of the    RNAi agent.-   13. The composition of embodiment 1, wherein the RNAi agent is    ligated to one or more diagnostic compound, reporter group,    cross-linking agent, nuclease-resistance conferring moiety, natural    or unusual nucleobase, lipophilic molecule, cholesterol, lipid,    lectin, steroid, uvaol, hecigenin, diosgenin, terpene, triterpene,    sarsasapogenin, Friedelin, epifriedelanol-derivatized lithocholic    acid, vitamin, carbohydrate, dextran, pullulan, chitin, chitosan,    synthetic carbohydrate, oligo lactate 15-mer, natural polymer, low-    or medium-molecular weight polymer, inulin, cyclodextrin, hyaluronic    acid, protein, protein-binding agent, integrin-targeting molecule,    polycationic, peptide, polyamine, peptide mimic, and/or transferrin.-   14. The composition of embodiment 1, wherein the RNAi agent is    capable of inhibiting expression of EPAS1 by at least about 50% in    786-O tumors in nude mice.-   15. The composition of embodiment 1, wherein the RNAi agent is    capable of inhibiting expression of EPAS1 by at least about 70% at a    concentration of 10 nM in 786-O cells in vitro.-   16. The composition of embodiment 1, wherein the RNAi agent is    capable of inhibiting expression of EPAS1 by at least about 80% at a    concentration of 10 nM in 786-O cells in vitro.-   17. The composition of embodiment 1, wherein the RNAi agent is    capable of inhibiting expression of EPAS1 by at least about 90% at a    concentration of 10 nM in 786-O cells in vitro.-   18. The composition of embodiment 1, wherein the RNAi has an EC50 of    no more than about 0.1 nM.-   19. The composition of embodiment 1, wherein the RNAi has an EC50 of    no more than about 0.01 nM.-   20. The composition of embodiment 1, wherein the RNAi has an EC50 of    no more than about 0.001 nM.-   21. A composition comprising an RNAi agent comprising a sense strand    and an antisense strand, wherein the sense strand and antisense    strand comprise at least 15 contiguous nucleotides differing by 0,    1, 2, or 3 nucleotides, from the sense and antisense strand,    respectively, of an RNAi agent to EPAS1 provided in any of Tables 1    to 5.-   22. The composition of embodiment 21, wherein the composition    comprises a second RNAi agent to EPAS1.-   23. The composition of embodiment 21, wherein the antisense strand    is 30 or fewer nucleotides in length.-   24. The composition of embodiment 21, wherein the sense strand and    the antisense strand form a duplex region 15 to 30 nucleotide pairs    in length.-   25. The composition of embodiment 21, wherein the antisense strand    and the sense strand are both 19 to 23 nucleotides in length.-   26. The composition of embodiment 21, wherein the RNAi agent    comprises a modification that causes the RNAi agent to have    increased stability in a biological sample or environment.-   27. The composition of embodiment 21 wherein the RNAi agent    comprises a modified sugar backbone, including a phosphorothioate    linkage, or a 2′-modified nucleotide.-   28. The composition of embodiment 21, wherein the RNAi agent    comprises: at least one 5′-uridine-adenine-3′ (5′-ua-3′)    dinucleotide, wherein the uridine is a 2′-modified nucleotide; at    least one 5′-uridine-guanine-3′ (5′-ug-3′) dinucleotide, wherein the    5′-uridine is a 2′-modified nucleotide; at least one    5′-cytidine-adenine-3′ (5′-ca-3′) dinucleotide, wherein the    5′-cytidine is a 2′-modified nucleotide; or at least one    5′-uridine-uridine-3′ (5′-uu-3′) dinucleotide, wherein the    5′-uridine is a 2′-modified nucleotide.-   29. The composition of embodiment 21, wherein the RNAi agent    comprises a 2′-modification selected from the group consisting of:    2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl    (2′-O-MOE), 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl    (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),    2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), and    2′-O—N-methylacetamido (2′-O-NMA).-   30. The composition of embodiment 21, wherein the RNAi agent    comprises a blunt end.-   31. The composition of embodiment 21, wherein the RNAi agent    comprises an overhang having 1 to 4 unpaired nucleotides.-   32. The composition of embodiment 21, wherein the RNAi agent    comprises an overhang at the 3′-end of the antisense strand of the    RNAi agent.-   33. The composition of embodiment 21, wherein the RNAi agent is    ligated to one or more diagnostic compound, reporter group,    cross-linking agent, nuclease-resistance conferring moiety, natural    or unusual nucleobase, lipophilic molecule, cholesterol, lipid,    lectin, steroid, uvaol, hecigenin, diosgenin, terpene, triterpene,    sarsasapogenin, Friedelin, epifriedelanol-derivatized lithocholic    acid, vitamin, carbohydrate, dextran, pullulan, chitin, chitosan,    synthetic carbohydrate, oligo lactate 15-mer, natural polymer, low-    or medium-molecular weight polymer, inulin, cyclodextrin, hyaluronic    acid, protein, protein-binding agent, integrin-targeting molecule,    polycationic, peptide, polyamine, peptide mimic, and/or transferrin.-   34. The composition of embodiment 21, wherein the RNAi agent is    capable of inhibiting expression of EPAS1 by at least about 50% in    786-O tumors in nude mice.-   35. The composition of embodiment 21, wherein the RNAi agent is    capable of inhibiting expression of EPAS1 by at least about 70% at a    concentration of 10 nM in 786-O cells in vitro.-   36. The composition of embodiment 21, wherein the RNAi agent is    capable of inhibiting expression of EPAS1 by at least about 80% at a    concentration of 10 nM in 786-O cells in vitro.-   37. The composition of embodiment 21, wherein the RNAi agent is    capable of inhibiting expression of EPAS1 by at least about 90% at a    concentration of 10 nM in 786-O cells in vitro.-   38. The composition of embodiment 21, wherein the RNAi has an EC50    of no more than about 0.1 nM.-   39. The composition of embodiment 21, wherein the RNAi has an EC50    of no mote than about 0.01 nM.-   40. The composition of embodiment 21, wherein the RNAi has an EC50    of no more than about 0.001 nM.-   41. A method of treating a EPAS1-related disease in an individual,    comprising the step of administering to the individual a    therapeutically effective amount of a composition comprising an RNAi    agent comprising a sense strand and an antisense strand, wherein the    antisense strand comprises at least 15 contiguous nucleotides    differing by 0, 1, 2, or 3 nucleotides front the antisense strand of    an RNAi agent to EPAS1 provided in any of Tables 1 to 5.-   42. The method of embodiment 41, wherein the EPAS1-related disease    is cancer, metastases, astrocytoma, bladder cancer, breast cancer,    chondrosarcoma, colorectal carcinoma, gastric carcinoma,    glioblastoma, head and neck squamous cell carcinoma, hepatocellular    carcinoma, lung adenocarcinoma, neuroblastoma, non-small cell lung    cancer, melanoma, multiple myeloma, ovarian cancer, rectal cancer,    renal cancer, clear cell renal cell carcinoma (and metastases of    this and other cancers), gingivitis, psoriasis, Kaposi's    sarcoma-associated herpesvirus, preeclampsia, inflammation, chronic    inflammation, neovascular diseases, or rheumatoid arthritis.-   43. The method of embodiment 41, wherein the EPAS1-related disease    is cancer.-   44. The method of embodiment 41, wherein the method further    comprises the step or administering additional treatment.-   45. The method of embodiment 44, wherein administration of the    composition comprising the RNAi agent and the additional treatment    is simultaneous, concurrent, separate or sequential.-   46. The method of embodiment 41, wherein the EPAS1-related disease    is a cancer, metastases, astrocytoma, bladder cancer, breast cancer,    chondrosarcoma, colorectal carcinoma, gastric carcinoma,    glioblastoma, head and neck squamous cell carcinoma, hepatocellular    carcinoma, lung adenocarcinoma, neuroblastoma, non-small cell lung    cancer, melanoma, multiple myeloma, ovarian cancer, rectal cancer,    renal cancer, clear cell renal cell carcinoma (and metastases of    this and other cancers), gingivitis, psoriasis, Kaposi's    sarcoma-associated herpesvirus, preeclampsia, inflammation, chronic    inflammation, neovascular diseases, or rheumatoid arthritis.-   47. The method of embodiment 41, where the EPAS1-related disease is    cancer.-   48. The method of embodiment 41, wherein the method further    comprises the step of administering an additional treatment.-   49. The method of embodiment 41, wherein administration of the    composition comprising the RNAi agent and the additional treatment    is simultaneous, concurrent, separate or sequential-   50. The method of embodiment 41, wherein the EPAS1-related disease    is cancer, metastases, astrocytoma, bladder cancer, breast cancer,    chondrosarcoma, colorectal carcinoma, gastric carcinoma,    glioblastoma, head and neck squamous cell carcinoma, hepatocellular    carcinoma, lung adenocarcinoma, neuroblastoma, non-small cell lung    cancer, melanoma, multiple myeloma, ovarian cancer, rectal cancer,    renal cancer, clear cell renal cell carcinoma (and metastases of    this and other cancers), gingivitis, psoriasis, Kaposi's    sarcoma-associated herpesvirus, preeclampsia, inflammation, chronic    inflammation, neovascular diseases, or rheumatoid arthritis.-   51. The method of embodiment 41, wherein the EPAS1-related disease    is cancer.-   52. The method of embodiment 41, wherein the composition comprises a    second RNAi agent to EPAS1.-   53. A method of inhibiting the expression of EPAS1 in an individual,    comprising the step of administering to the individual a    therapeutically effective amount of a composition comprising an RNAi    agent comprising a sense strand and an antisense strand, wherein the    antisense strand comprises at least 15 contiguous nucleotides    differing by 0, 1, 2, or 3 nucleotides from the antisense strand of    an RNAi agent to EPAS1 provided in any of Tables 1 to 5.-   54. The method of embodiment 53, wherein the individual is afflicted    with or susceptible to an EPAS1-related disease.-   55. The method of embodiment 53, wherein the EPAS1-related disease    is cancer, metastases, astrocytoma, bladder cancer, breast cancer,    chondrosarcoma, colorectal carcinoma, gastric carcinoma,    glioblastoma, head and neck squamous cell carcinoma, hepatocellular    carcinoma, lung adenocarcinoma, neuroblastoma, non-small cell lung    cancer, melanoma, multiple myeloma, ovarian cancer, rectal cancer,    renal cancer, clear cell renal cell carcinoma (and metastases of    this and other cancers), gingivitis, psoriasis, Kaposi's    sarcoma-associated herpesvirus, preeclampsia, inflammation, chronic    inflammation, neovascular diseases, or rheumatoid arthritis.-   56. The method of embodiment 53, wherein the EPAS1-related disease    is cancer-   57. The method of embodiment 53, wherein the method further    comprises the step of administering an additional disease treatment.-   58. The method of embodiment 53, wherein the method further    comprises the step of administering an additional disease treatment,    wherein administration of the composition comprising the RNAi agent    and the additional disease treatment is simultaneous, concurrent,    separate or sequential.-   59. The method of embodiment 53, wherein the EPAS1-related disease    is cancer, metastases, astrocytoma, bladder cancer, breast cancer,    chondrosarcoma, colorectal carcinoma, gastric carcinoma,    glioblastoma, head and neck squamous cell carcinoma, hepatocellular    carcinoma, lung adenocarcinoma, neuroblastoma, non-small cell lung    cancer, melanoma, multiple myeloma, ovarian cancer, rectal cancer,    renal cancer, clear cell renal cell carcinoma (and metastases of    this and other cancers), gingivitis, psoriasis, Kaposi's    sarcoma-associated herpesvirus, preeclampsia, inflammation, chronic    inflammation, neovascular diseases, or rheumatoid arthritis.-   60. The method of embodiment 53, where the EPAS1-related disease is    cancer-   61. The method of embodiment 53, wherein the method further    comprises the step of administering an additional disease treatment.-   62. The method of embodiment 53, wherein the method further    comprises the step of administering an additional disease treatment,    wherein administration of the composition comprising the RNAi agent    and the additional disease treatment is simultaneous, concurrent,    separate or sequential.-   63. The method of embodiment 53, wherein the EPAS1-relate disease is    cancer, metastases, astrocytoma, bladder cancer, breast cancer,    chondrosarcoma, colorectal carcinoma, gastric carcinoma,    glioblastoma, head and neck squamous cell carcinoma, hepatocellular    carcinoma, lung adenocarcinoma, neuroblastoma, non-small cell lung    cancer, melanoma, multiple myeloma, ovarian cancer, rectal cancer,    renal cancer, clear cell renal cell carcinoma (and metastases of    this and other cancers), gingivitis, psoriasis, Kaposi's    sarcoma-associated herpesvirus, preeclampsia, inflammation, chronic    inflammation, neovascular diseases, or rheumatoid arthritis.-   64. The method of embodiment 53, wherein the EPAS1-related disease    is cancer.-   65. The method of embodiment 53, wherein the composition further    comprises a second RNAi agent to EPAS1.-   66. The composition according to embodiment 1 or embodiment 21, for    use in a method of treating a EPAS1-related disease in an    individual, the method comprising the step of administering to the    individual a therapeutically effective amount of a composition    according to embodiment 1 or embodiment 21.-   67. The composition according to embodiment 1 or embodiment 21, for    use in a method of inhibiting the expression of EPAS1 in an    individual, the method comprising the step of administering to the    individual a therapeutically effective amount of a composition    according to embodiment 1 or embodiment 21.-   68. The use of a composition according to embodiment 1 or embodiment    21, in the manufacture of a medicament for treatment of an    EPAS1-related disease.-   69. The use of embodiment 68, wherein the EPAS1-related disease is    cancer, metastases, astrocytoma, bladder cancer, breast cancer,    chondrosarcoma, colorectal carcinoma, gastric carcinoma,    glioblastoma, head and neck squamous cell carcinoma, hepatocellular    carcinoma, lung adenocarcinoma, neuroblastoma, non-small cell lung    cancer, melanoma, multiple myeloma, ovarian cancer, rectal cancer,    renal cancer, clear cell renal cell carcinoma (and metastases of    this and other cancers), gingivitis, psoriasis, Kaposi's    sarcoma-associated herpesvirus, preeclampsia, inflammation, chronic    inflammation, neovascular diseases, or rheumatoid arthritis.-   70. The composition of embodiment 1 or embodiment 21, for use in the    treatment of an EPAS1-related disease.-   71. The use of embodiment 70, wherein the EPAS1-related disease is    cancer-   72. A method of inhibiting the expression of EPAS1 in an cell,    comprising the step of introducing into the cell a composition    comprising an RNAi agent comprising a sense strand and an antisense    strand, wherein the antisense strand comprises at least 15    contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from    the antisense strand of an RNAi agent to EPAS1 provided in any of    Tables 1 to 5.-   73. The composition of embodiment 1, wherein all pyrimidines are    2′-O-methyl-modified nucleotides.-   74. The composition of embodiment 21, wherein all pyrimidines are    2′-O-methyl-modified nucleotides.

Unless defined otherwise, the technical and scientific terms used herein have the same meaning as that usually understood by a specialistfamiliar with the field to which the disclosure belongs.

Unless indicated otherwise, all methods, steps, techniques andmanipulations that are not specifically described in detail can beperformed and have been performed in a manner known per se, as will beclear to the skilled person. Reference is for example again made to thestandard handbooks and the general background art mentioned herein andto the further references cited therein. Unless indicated otherwise,each of the references cited herein is incorporated in its entirety byreference.

Claims to the invention are non-limiting and are provided below.

It is also noted that where the Claims recite a particular SEQ ID NO,claimed molecules of the recited sequence encompass molecules which arenot modified or are modified by any known modification (e.g., with orwithout 2′-modifications, terminal dinucleotides, 3′ end caps, etc.),unless the Claims recite otherwise. Although particular aspects andclaims have been, disclosed herein in detail, this has been done by wayof example for purposes of illustration only, and is not intended to belimiting with respect to the scope of the appended claims, or the scopeof subject matter of claims of any corresponding future application. Inparticular, it is contemplated by the inventors that varioussubstitutions, alterations, and modifications may be made to thedisclosure without departing from the spirit and scope of the disclosureas defined by the claims. The choice of nucleic acid starting material,clone of interest, or library type is believed to be a matter of routinefor a person of ordinary skill in the art with knowledge of the aspectsdescribed herein. Other aspects, advantages, and modificationsconsidered to be within the scope of the following claims. Those skilledin the art will recognize or be able to ascertain, using no more thanroutine experimentation, many equivalents of the specific aspects of theinvention described herein. Such equivalents are intended to beencompassed by the following claims. Redrafting of claim scope in laterfiled corresponding applications may be due to limitations by the patentlaws of various countries and should not be interpreted as giving upsubject matter of the claims.

We claim:
 1. A method of treating a EPAS1-related disease in anindividual, comprising the step of administering to the individual atherapeutically effective amount of a composition comprising an RNAiagent comprising a sense strand and an antisense strand, wherein theantisense strand comprises nucleotides 2-18 of the nucleotide sequenceof SEQ ID NO: 72, and the sense strand comprises the nucleotide sequenceSEQ ID NO: 53 differing by 0, 1, 2, or 3 nucleotides, wherein the sensestrand is between 19 and 30 nucleotides in length and the antisensestrand is between 18 and 30 nucleotides in length, and at least onenucleotide is a modified nucleotide.
 2. The method of claim 1, whereinthe individual is afflicted with or susceptible to an EPAS1-relateddisease.
 3. The method of claim 1, wherein the EPAS1-related disease iscancer, metastases, astrocytoma, bladder cancer, breast cancer,chondrosarcoma, colorectal carcinoma, gastric carcinoma, glioblastoma,head and neck squamous cell carcinoma, hepatocellular carcinoma, lungadenocarcinoma, neuroblastoma, non-small cell lung cancer, melanoma,multiple myeloma, ovarian cancer, rectal cancer, renal cancer, clearcell renal cell carcinoma (and metastases of this and other cancers),gingivitis, psoriasis, Kaposi's sarcoma-associated herpesvirus,preeclampsia, inflammation, chronic inflammation, neovascular diseases,or rheumatoid arthritis.
 4. The method of claim 1, wherein theEPAS1-related disease is cancer.
 5. The method of claim 1, wherein themethod further comprises the step of administering an additional diseasetreatment.
 6. The method of claim 1, wherein the composition furthercomprises a second RNAi agent to EPAS1.
 7. The method of claim 1,wherein the antisense strand comprises the nucleotide sequence of SEQ IDNO: 72, and the sense strand comprises the nucleotide sequence of SEQ IDNO: 53 differing by 0, 1, 2, or 3 nucleotides.
 8. The method of claim 1,wherein the antisense strand comprises the nucleotide sequence of SEQ IDNO: 72, and the sense strand comprises the nucleotide sequence of SEQ IDNO: 53 differing by 0, 1, or 2 nucleotides.
 9. The method of claim 1,wherein the antisense strand comprises the nucleotide sequence of SEQ IDNO: 72, and the sense strand comprises the nucleotide sequence of SEQ IDNO: 53 differing by 0, or 1 nucleotides.
 10. The method of claim 1,wherein the antisense strand comprises the nucleotide sequence of SEQ IDNO: 72, and the sense strand comprises the nucleotide sequence of SEQ IDNO:
 53. 11. The method of claim 1, wherein the antisense strand is about23 or fewer nucleotides in length.
 12. The method of claim 1, whereinthe sense strand and the antisense strand form a duplex region about 15to about 30 nucleotide pairs in length.
 13. The method of claim 1,wherein the antisense strand and the sense strand are both about 19 toabout 23 nucleotides in length.
 14. The method of claim 1, wherein theRNAi agent comprises a 2′-modification selected from the groupconsisting of: 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-methyl,2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-ODMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-ODMAEOE), and2′-ON-methylacetamido (2′-O-NMA).
 15. The method of claim 1, wherein theRNAi agent is ligated to one or more diagnostic compound, reportergroup, cross-linking agent, nuclease-resistance conferring moiety,natural or unusual nucleobase, lipophilic molecule, cholesterol, lipid,lectin, steroid, uvaol, hecogenin, diosgenin, terpene, triterpene,sarsasapogenin, Friedelin, epifriedelanol-derivatized lithocholic acid,vitamin, carbohydrate, dextran, pullulan, chitin, chitosan, syntheticcarbohydrate, oligo lactate 15-mer, natural polymer, low- ormedium-molecular weight polymer, inulin, cyclodextrin, hyaluronic acid,protein, protein-binding agent, integrin-targeting molecule,polycationic, peptide, polyamine, peptide mimic, and transferrin. 16.The method of claim 1, wherein the antisense strand comprises themodified nucleotide sequence of SEQ ID NO: 110, and the sense strandcomprises the modified nucleotide sequence of SEQ ID NO:
 91. 17. Themethod of claim 1, wherein the antisense strand comprises the modifiednucleotide sequence of SEQ ID NO: 140, and the sense strand comprisesthe modified nucleotide sequence of SEQ ID NO: 159.